Mice expressing a limited immunoglobulin light chain repertoire

ABSTRACT

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that present a choice of two human light chain variable gene segments such that the immunoglobulin light chains expresses by the mouse comprise one of the two human light chain variable gene segments. Methods for making bispecific antibodies having universal light chains using mice as described herein, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, and host cells are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 13/488,628filed Jun. 5, 2012, which is a continuation-in-part of U.S. Ser. No.13/412,936 filed Mar. 6, 2012, which is a continuation-in-part of U.S.Ser. No. 13/093,156 filed Apr. 25, 2011, which is a continuation-in-partof U.S. Ser. No. 13/022,759 filed Feb. 8, 2011, which is anonprovisional application of U.S. Provisional Application Ser. No.61/302,282, filed Feb. 8, 2010; which applications are herebyincorporated by reference in their entirety.

FIELD

A genetically modified mouse is provided that expresses antibodieshaving a common human variable/mouse constant light chain associatedwith diverse human variable/mouse constant heavy chains. A method formaking a human bispecific antibody from human variable region genesequences of B cells of the mouse is provided.

BACKGROUND

Antibodies typically comprise a homodimeric heavy chain component,wherein each heavy chain monomer is associated with an identical lightchain. Antibodies having a heterodimeric heavy chain component (e.g.,bispecific antibodies) are desirable as therapeutic antibodies. Butmaking bispecific antibodies having a suitable light chain componentthat can satisfactorily associate with each of the heavy chains of abispecific antibody has proved problematic.

In one approach, a light chain might be selected by surveying usagestatistics for all light chain variable domains, identifying the mostfrequently employed light chain in human antibodies, and pairing thatlight chain in vitro with the two heavy chains of differing specificity.

In another approach, a light chain might be selected by observing lightchain sequences in a phage display library (e.g., a phage displaylibrary comprising human light chain variable region sequences, e.g., ahuman scFv library) and selecting the most commonly used light chainvariable region from the library. The light chain can then be tested onthe two different heavy chains of interest.

In another approach, a light chain might be selected by assaying a phagedisplay library of light chain variable sequences using the heavy chainvariable sequences of both heavy chains of interest as probes. A lightchain that associates with both heavy chain variable sequences might beselected as a light chain for the heavy chains.

In another approach, a candidate light chain might be aligned with theheavy chains' cognate light chains, and modifications are made in thelight chain to more closely match sequence characteristics common to thecognate light chains of both heavy chains. If the chances ofimmunogenicity need to be minimized, the modifications preferably resultin sequences that are present in known human light chain sequences, suchthat proteolytic processing is unlikely to generate a T cell epitopebased on parameters and methods known in the art for assessing thelikelihood of immunogenicity (i.e., in silico as well as wet assays).

All of the above approaches rely on in vitro methods that subsume anumber of a priori restraints, e.g., sequence identity, ability toassociate with specific pre-selected heavy chains, etc. There is a needin the art for compositions and methods that do not rely on manipulatingin vitro conditions, but that instead employ more biologically sensibleapproaches to making human epitope-binding proteins that include acommon light chain.

SUMMARY

Genetically modified mice that express human immunoglobulin heavy andlight chain variable domains, wherein the mice have a limited lightchain variable repertoire, are provided. A biological system forgenerating a human light chain variable domain that associates andexpresses with a diverse repertoire of affinity-matured human heavychain variable domains is provided. Methods for making binding proteinscomprising immunoglobulin variable domains are provided, comprisingimmunizing mice that have a limited immunoglobulin light chainrepertoire with an antigen of interest, and employing an immunoglobulinvariable region gene sequence of the mouse in a binding protein thatspecifically binds the antigen of interest. Methods include methods formaking human immunoglobulin heavy chain variable domains suitable foruse in making multi-specific antigen-binding proteins.

Genetically engineered mice are provided that select suitableaffinity-matured human immunoglobulin heavy chain variable domainsderived from a repertoire of unrearranged human heavy chain variableregion gene segments, wherein the affinity-matured human heavy chainvariable domains associate and express with a single human light chainvariable domain derived from one human light chain variable region genesegment. Genetically engineered mice that present a choice of two humanlight chain variable region gene segments are also provided. In variousaspects, the one or two gene segments include human Vκ1-39 and/or humanVκ3-20.

Genetically engineered mice are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region gene segments. In some embodiments, provided miceare genetically engineered to include a single unrearranged human lightchain variable region gene segment (or two human light chain variableregion gene segments) that rearranges to form a rearranged human lightchain variable region gene (or two rearranged light chain variableregion genes) that expresses a single light chain (or that expresseither or both of two light chains). The rearranged human light chainvariable domains are capable of pairing with a plurality ofaffinity-matured human heavy chains selected by the mice, wherein theheavy chain variable regions specifically bind different epitopes.

Genetically engineered mice are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region sequences. In some embodiments, provided mice aregenetically engineered to include a single V/J human light chainsequence (or two V/J sequences) that express a variable region of asingle light chain (or that express either or both of two variableregions). A light chain comprising the variable sequence is capable ofpairing with a plurality of affinity-matured human heavy chains clonallyselected by the mice, wherein the heavy chain variable regionsspecifically bind different epitopes.

In one aspect, a genetically modified mouse is provided that comprises asingle human immunoglobulin light chain variable (V_(L)) region genesegment that is capable of rearranging with a human J gene segment(selected from one or a plurality of J_(L) segments) and encoding ahuman V_(L) domain of an immunoglobulin light chain. In another aspect,a genetically modified mouse is provided that comprises no more than twohuman V_(L) gene segments, each of which is capable of rearranging witha human J gene segment (selected from one or a plurality of J_(L)segments) and encoding a human V_(L) domain of an immunoglobulin lightchain. In some embodiments, the two human V_(L) gene segments arejuxtaposed in the genome of the mouse. In some embodiments, the twohuman V_(L) gene segments are at different loci (e.g., a heterozygote,comprising a first human V_(L) segment at a first light chain allele,and a second human V_(L) segment at a second light chain allele, whereinthe first and the second human V_(L) segments are not identical) in thegenome of the mouse. In some embodiments, the two human V_(L) genesegments are a human Vκ1-39 gene segment and a human Vκ3-20 genesegment. In one embodiment, the human J_(L) gene segment is selectedfrom the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and pairwisecombinations thereof. In various embodiments, a provided geneticallyengineered mouse is incapable of expressing an immunoglobulin lightchain that contains an endogenous V_(L) gene segment. For example, insome embodiments, a provided genetically engineered mouse contains agenetic modification that inactivates and/or removes part or all of anendogenous V_(L) gene segment.

In one embodiment, the single human V_(L) gene segment is operablylinked to a human J_(L) gene segment selected from Jκ1, Jκ2, Jκ3, Jκ4,and Jκ5, wherein the single human V_(L) gene segment is capable ofrearranging to form a sequence encoding a light chain variable regiongene with any of the one or more human J_(L) gene segments.

In one embodiment, a provided genetically modified mouse comprises animmunoglobulin light chain locus that does not comprise an endogenousmouse V_(L) gene segment that is capable of rearranging to form animmunoglobulin light chain gene, wherein the V_(L) locus contains asingle human V_(L) gene segment that is capable of rearranging to encodea V_(L) region of a light chain gene. In specific embodiments, the humanV_(L) gene segment is a human Vκ1-39Jκ5 gene segment or a humanVκ3-20Jκ1 gene segment. In some embodiments, a provided geneticallymodified mouse comprises a V_(L) locus that does not comprise anendogenous mouse V_(L) gene segment that is capable of rearranging toform an immunoglobulin light chain gene, wherein the V_(L) locuscomprises no more than two human V_(L) gene segments that are capable ofrearranging to encode a V_(L) region of a light chain gene. In somecertain embodiments, the no more than two human V_(L) gene segments areselected from the group consisting of a human Vκ1-39 gene segment, ahuman Vκ3-20 gene segment, and a combination thereof. In some certainembodiments, the no more than two human V_(L) gene segments are a humanVκ1-39Jκ5 gene segment and a human Vκ3-20Jκ1 gene segment.

In one aspect, a genetically modified mouse is provided that comprises asingle rearranged (V/J) human immunoglobulin light chain variable(V_(L)) region (i.e., a V_(L)/J_(L) region) that encodes a human V_(L)domain of an immunoglobulin light chain. In another aspect, the mousecomprises no more than two rearranged human V_(L) regions that arecapable of encoding a human V_(L) domain of an immunoglobulin lightchain.

In one embodiment, the V_(L) region is a rearranged human Vκ1-39/Jsequence or a rearranged human Vκ3-20/J sequence. In one embodiment, thehuman J_(L) segment of the rearranged V_(L)/J_(L) sequence is selectedfrom Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In a specific embodiment, the V_(L)region is a human Vκ1-39Jκ5 sequence or a human Vκ3-20Jκ1 sequence. In aspecific embodiment, the mouse has both a human Vκ1-39Jκ5 sequence and ahuman Vκ3-20Jκ1 sequence.

In one embodiment, the human V_(L) gene segment is operably linked to ahuman or mouse leader sequence. In one embodiment, the leader sequenceis a mouse leader sequence. In a specific embodiment, the mouse leadersequence is a mouse Vκ3-7 leader sequence. In a specific embodiment, theleader sequence is operably linked to an unrearranged human V_(L) genesegment. In a specific embodiment, the leader sequence is operablylinked to a rearranged human V_(L)/J_(L) sequence.

In one embodiment, the V_(L) gene segment is operably linked to animmunoglobulin promoter sequence. In one embodiment, the promotersequence is a human promoter sequence. In a specific embodiment, thehuman immunoglobulin promoter is a human Vκ3-15 promoter. In a specificembodiment, the promoter is operably linked to an unrearranged humanV_(L) gene segment. In a specific embodiment, the promoter is operablylinked to a rearranged human V_(L)/J_(L) sequence.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with ahuman V_(L) gene segment that rearranges with a human J segment andencodes a V_(L) domain of a reverse chimeric light chain comprising anendogenous mouse light chain constant region (C_(L)). In a specificembodiment, the V_(L) gene segment is at the mouse Vκ locus, and themouse C_(L) is a mouse Cκ.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with arearranged human V_(L) region (V_(L)/J_(L) sequence) and encodes a V_(L)domain of a reverse chimeric light chain comprising an endogenous mouselight chain constant region (C_(L)). In a specific embodiment, therearranged human V_(L)/J_(L) sequence is at the mouse kappa (κ) locus,and the mouse C_(L) is a mouse Cκ.

In one embodiment, the V_(L) locus of the modified mouse is a κ lightchain locus, and the κ light chain locus comprises a mouse κ intronicenhancer, a mouse κ 3′ enhancer, or both an intronic enhancer and a 3′enhancer.

In one embodiment, the mouse comprises a nonfunctional immunoglobulinlambda (λ) light chain locus. In a specific embodiment, the λ lightchain locus comprises a deletion of one or more sequences of the locus,wherein the one or more deletions renders the λ light chain locusincapable of rearranging to form a light chain gene. In anotherembodiment, all or substantially all of the V_(L) gene segments of the λlight chain locus are deleted.

In one embodiment, mouse makes a light chain that comprises asomatically mutated V_(L) domain derived from a human V_(L) genesegment. In one embodiment, the light chain comprises a somaticallymutated V_(L) domain derived from a human V_(L) gene segment, and amouse Cκ region. In one embodiment, the mouse does not express a λ lightchain.

In one embodiment, the genetically modified mouse is capable ofsomatically hypermutating the human V_(L) region sequence. In a specificembodiment, the mouse comprises a cell that comprises a rearrangedimmunoglobulin light chain gene derived from a human V_(L) gene segmentthat is capable of rearranging and encoding a V_(L) domain, and therearranged immunoglobulin light chain gene comprises a somaticallymutated V_(L) domain.

In one embodiment, the mouse comprises a cell that expresses a lightchain comprising a somatically mutated human V_(L) domain linked to amouse Cκ, wherein the light chain associates with a heavy chaincomprising a somatically mutated V_(H) domain derived from a human V_(H)gene segment and wherein the heavy chain comprises a mouse heavy chainconstant region (C_(H)). In a specific embodiment, the heavy chaincomprises a mouse C_(H)1, a mouse hinge, a mouse C_(H)2, and a mouseC_(H)3. In a specific embodiment, the heavy chain comprises a humanC_(H)1, a hinge, a mouse C_(H)2, and a mouse C_(H)3.

In one embodiment, the mouse comprises a replacement of endogenous mouseV_(H) gene segments with one or more human V_(H) gene segments, whereinthe human V_(H) gene segments are operably linked to a mouse C_(H)region gene, such that the mouse rearranges the human V_(H) genesegments and expresses a reverse chimeric immunoglobulin heavy chainthat comprises a human V_(H) domain and a mouse C_(H). In oneembodiment, 90-100% of unrearranged mouse V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In aspecific embodiment, all or substantially all of the endogenous mouseV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In one embodiment, the replacement is with at least19, at least 39, or at least 80 or 81 unrearranged human V_(H) genesegments. In one embodiment, the replacement is with at least 12functional unrearranged human V_(H) gene segments, at least 25functional unrearranged human V_(H) gene segments, or at least 43functional unrearranged human V_(H) gene segments. In one embodiment,the mouse comprises a replacement of all mouse D_(H) and J_(H) segmentswith at least one unrearranged human D_(H) segment and at least oneunrearranged human J_(H) segment. In one embodiment, the at least oneunrearranged human D_(H) segment is selected from 1-1, D1-7, 1-26, 2-8,2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13, 7-27, and acombination thereof. In one embodiment, the at least one unrearrangedhuman J_(H) segment is selected from 1, 2, 3, 4, 5, 6, and a combinationthereof. In a specific embodiment, the one or more human V_(H) genesegment is selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11,3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51,a 6-1 human V_(H) gene segment, and a combination thereof.

In one embodiment, the mouse comprises a B cell that expresses a bindingprotein that specifically binds an antigen of interest, wherein thebinding protein comprises a light chain derived from a human Vκ1-39/Jκ5rearrangement or a human Vκ3-20/Jκ1 rearrangement, and wherein the cellcomprises a rearranged immunoglobulin heavy chain gene derived from arearrangement of human V_(H) gene segments selected from a 1-69, 2-5,3-13, 3-23, 3-30, 3-33, 3-53, 4-39, 4-59, and 5-51 gene segment. In oneembodiment, the one or more human V_(H) gene segments are rearrangedwith a human heavy chain J_(H) gene segment selected from 1, 2, 3, 4, 5,and 6. In one embodiment, the one or more human V_(H) and J_(H) genesegments are rearranged with a human D_(H) gene segment selected from1-1, 1-7, 1-26, 2-8, 2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13,and 7-27. In a specific embodiment, the light chain gene has 1, 2, 3, 4,or 5 or more somatic hypermutations.

In one embodiment, the mouse comprises a B cell that comprises arearranged immunoglobulin heavy chain variable region gene sequencecomprising a V_(H)/D_(H)/J_(H) region selected from 2-5/6-6/1,2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4,3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/3, 3-30/3-3/4,3-30/3-22/5, 3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3,3-30/6-6/4, 3-30/6-6/5, 3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5,3-30/7-27/6, 3-33/1-7/4, 3-33/2-15/4, 4-39/1-26/3, 4-59/3-16/3,4-59/3-16/4, 4-59/3-22/3, 5-51/3-16/6, 5-51/5-5/3, 5-51/6-13/5,3-53/1-1/4, 1-69/6-6/5, and 1-69/6-13/4. In a specific embodiment, the Bcell expresses a binding protein comprising a human immunoglobulin heavychain variable region fused with a mouse heavy chain constant region,and a human immunoglobulin light chain variable region fused with amouse light chain constant region.

In one embodiment, the rearranged human V_(L) region is a humanVκ1-39Jκ5 sequence, and the mouse expresses a reverse chimeric lightchain comprising (i) a V_(L) domain derived from the human V_(L)/J_(L)sequence and (ii) a mouse C_(L); wherein the light chain is associatedwith a reverse chimeric heavy chain comprising (i) a mouse C_(H) and(ii) a somatically mutated human V_(H) domain derived from a human V_(H)gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11,3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51,a 6-1 human V_(H) gene segment, and a combination thereof. In oneembodiment, the mouse expresses a light chain that is somaticallymutated. In one embodiment the C_(L) is a mouse Cκ. In a specificembodiment, the human V_(H) gene segment is selected from a 2-5, 3-13,3-23, 3-30, 4-59, 5-51, and 1-69 gene segment. In a specific embodiment,the somatically mutated human V_(H) domain comprises a sequence derivedfrom a D_(H) segment selected from 1-1, 1-7, 2-8, 3-3, 3-10, 3-16, 3-22,5-5, 5-12, 6-6, 6-13, and 7-27. In a specific embodiment, thesomatically mutated human V_(H) domain comprises a sequence derived froma J_(H) segment selected from 1, 2, 3, 4, 5, and 6. In a specificembodiment, the somatically mutated human V_(H) domain is encoded by arearranged human V_(H)/D_(H)/J_(H) sequence selected from 2-5/6-6/1,2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4,3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/4, 3-30/3-22/5,3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3, 3-30/6-6/4, 3-30/6-6/5,3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5, 3-30/7-27/6, 4-59/3-16/3,4-59/3-16/4, 4-59/3-22/3, 5-51/5-5/3, 1-69/6-6/5, and 1-69/6-13/4.

In one embodiment, the rearranged human V_(L) region is a humanVκ3-20Jκ1 sequence, and the mouse expresses a reverse chimeric lightchain comprising (i) a V_(L) domain derived from the rearranged humanV_(L)/J_(L) sequence, and (ii) a mouse C_(L); wherein the light chain isassociated with a reverse chimeric heavy chain comprising (i) a mouseC_(H), and (ii) a somatically mutated human V_(H) derived from a humanV_(H) gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9,3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59,5-51, a 6-1 human V_(H) gene segment, and a combination thereof. In oneembodiment, the mouse expresses a light chain that is somaticallymutated. In one embodiment the C_(L) is a mouse Cκ. In a specificembodiment, the human V_(H) gene segment is selected from a 3-30, 3-33,3-53, 4-39, and 5-51 gene segment. In a specific embodiment, thesomatically mutated human V_(H) domain comprises a sequence derived froma D_(H) segment selected from 1-1, 1-7, 1-26, 2-15, 3-3, 3-16, and 6-13.In a specific embodiment, the somatically mutated human V_(H) domaincomprises a sequence derived from a J_(H) segment selected from 3, 4, 5,and 6. In a specific embodiment, the somatically mutated human V_(H)domain is encoded by a rearranged human V_(H)/D_(H)/J_(H) sequenceselected from 3-30/1-1/4, 3-30/3-3/3, 3-33/1-7/4, 3-33/2-15/4,4-39/1-26/3, 5-51/3-16/6, 5-51/6-13/5, and 3-53/1-1/4.

In one embodiment, the mouse comprises both a rearranged human Vκ1-39Jκ5sequence and a rearranged human Vκ3-20Jκ1 sequence, and the mouseexpresses a reverse chimeric light chain comprising (i) a V_(L) domainderived from the human Vκ1-39Jκ5 sequence or the human Vκ3-20Jκ1sequence, and (ii) a mouse C_(L); wherein the light chain is associatedwith a reverse chimeric heavy chain comprising (i) a mouse C_(H), and(ii) a somatically mutated human V_(H) derived from a human V_(H) genesegment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13,3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a 6-1human V_(H) gene segment, and a combination thereof. In one embodiment,the mouse expresses a light chain that is somatically mutated. In oneembodiment the C_(L) is a mouse Cκ.

In one embodiment, 90-100% of the endogenous unrearranged mouse V_(H)gene segments are replaced with at least one unrearranged human V_(H)gene segment. In a specific embodiment, all or substantially all of theendogenous unrearranged mouse V_(H) gene segments are replaced with atleast one unrearranged human V_(H) gene segment. In one embodiment, thereplacement is with at least 18, at least 39, at least 80, or 81unrearranged human V_(H) gene segments. In one embodiment, thereplacement is with at least 12 functional unrearranged human V_(H) genesegments, at least 25 functional unrearranged human V_(H) gene segments,or at least 43 unrearranged human V_(H) gene segments.

In one embodiment, the genetically modified mouse is a C57BL strain, ina specific embodiment selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In a specific embodiment, thegenetically modified mouse is a mix of an aforementioned 129 strain andan aforementioned C57BL/6 strain. In another specific embodiment, themouse is a mix of aforementioned 129 strains, or a mix of aforementionedBL/6 strains. In a specific embodiment, the 129 strain of the mix is a129S6 (129/SvEvTac) strain.

In one embodiment, the mouse expresses a reverse chimeric antibodycomprising a light chain that comprises a mouse Cκ and a somaticallymutated human V_(L) domain derived from a rearranged human Vκ1-39Jκ5sequence or a rearranged human Vκ3-20Jκ1 sequence, and a heavy chainthat comprises a mouse C_(H) and a somatically mutated human V_(H)domain derived from a human V_(H) gene segment selected from a 1-2, 1-8,1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33,3-48, 3-53, 4-31, 4-39, 4-59, 5-51, and a 6-1 human V_(H) gene segment,wherein the mouse does not express a fully mouse antibody and does notexpress a fully human antibody. In one embodiment the mouse comprises aκ light chain locus that comprises a replacement of endogenous mouse κlight chain gene segments with the rearranged human Vκ1-39Jκ5 sequenceor the rearranged human Vκ3-20Jκ1 sequence, and comprises a replacementof all or substantially all endogenous mouse V_(H) gene segments with acomplete or substantially complete repertoire of human V_(H) genesegments.

In one aspect, a population of antigen-specific antibodies derived froma mouse as described herein is provided, wherein the antibodies comprisea light chain gene derived from a human Vκ1-39/Jκ5 rearrangement or ahuman Vκ3-20/Jκ1 rearrangement, and wherein the antibodies comprise arearranged immunoglobulin heavy chain gene derived from a rearrangementof a human V_(H) gene segment selected from a 1-2, 1-3, 1-8, 1-18, 1-24,1-46, 1-58, 1-69, 2-5, 2-26, 2-70, 3-7, 3-9, 3-11, 3-13, 3-15, 3-16,3-20, 3-21, 3-23, 3-30, 3-33, 3-43, 3-48, 3-53, 3-64, 3-72, 3-73, 4-31,4-34, 4-39, 4-59, 5-51, and a 6-1 human V_(H) gene segment. In oneembodiment, the one or more human V_(H) gene segments are rearrangedwith a human heavy chain J_(H) gene segment selected from 1, 2, 3, 4, 5,and 6. In a specific embodiment, the light chain has 1, 2, 3, 4, or 5 ormore somatic hypermutations.

In one embodiment, the light chain has 1, 2, 3, or 4 somatichypermutations. In one embodiment, the light chain gene has 1 or 2mutations. In various embodiments, the light chain gene is capable ofincurring multiple mutations along its sequence.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and the light chain has at least one or no more than foursomatic hypermutations. In one embodiment, the light chain comprises atleast two somatic hypermutations. In one embodiment, the light chaincomprises at least three somatic hypermutations. In one embodiment, thelight chain comprises at least four somatic hypermutations. In aspecific embodiment, at least one such somatic hypermutation is presentin one or more framework regions (FWs) of the light chain. In a specificembodiment, at least one such somatic hypermutation is present in one ormore complementarity determining regions (CDRs) of the light chain. In aspecific embodiment, at least one such somatic hypermutation is presentin one or more FWs and/or one or more CDRs of the light chain. Invarious embodiments, the framework regions are selected from framework 1(FW1), framework 2 (FW2), framework 3 (FW3), and/or a combinationthereof. In various embodiments, the CDRs are selected from CDR1, CDR2,CDR3, and/or a combination thereof.

In one embodiment, the heavy chain comprises at least one mutation inone or more FWs or one or more CDRs. In one embodiment, the heavy chaincomprises at least one mutation in one or more FWs and one or more CDRs.In one embodiment, the heavy chain comprises at least two mutations inone or more FWs and one or more CDRs. In one embodiment, the heavy chaincomprises at least three mutations in one or more FWs and one or moreCDRs. In one embodiment, the heavy chain comprises at least fourmutations in one or more FWs and one or more CDRs. In one embodiment,the heavy chain comprises at least five or more than five mutations inone or more FWs and one or more CDRs; in a specific embodiment, theheavy chain comprises at least five or more than five mutations in twoFWs; in a specific embodiment, the heavy chain comprises at least fiveor more than five mutations in one FW and one CDR.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 9% of the Vκ1-39/Jκ5-derived light chains haveat least one mutation present in FW1; in one embodiment, at least 9% ofthe light chains comprise one mutation present in FW1. In oneembodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 25% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than two mutations present in CDR1; in oneembodiment, at least 19% of the light chains have one mutation presentin CDR1; in one embodiment, at least 5% of the light chains have twomutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 20% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than three mutations present in FW2; in oneembodiment, at least 17% of the light chains have one mutation presentin FW2; in one embodiment, at least 1% of the light chains have twomutations present in FW2; in one embodiment, at least 1% of the lightchains have three mutations present in FW2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 10% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than two mutations present in CDR2; in oneembodiment, at least 10% of the light chains have one mutation presentin CDR2; in one embodiment, at least 1% of the light chains have twomutations present in CDR2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 29% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than four mutations present in FW3; in oneembodiment, at least 21% of the light chains have one mutation presentin FW3; in one embodiment, at least 5% of the light chains have twomutations present in FW3; in one embodiment, at least 2% of the lightchains have three mutations present in FW3; in one embodiment, at least2% of the light chains have four mutations present in FW3.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 37% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than four mutations present in CDR3; in oneembodiment, at least 27% of the light chains have one mutation presentin CDR3; in one embodiment, at least 8% of the light chains have twomutations present in CDR3; in one embodiment, at least 1% of the lightchains have three mutations present in CDR3; in one embodiment, at least1% of the light chains have four mutations present in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ1-39/Jκ5 rearrangement andabout 9% of the Vκ1-39/Jκ5-derived light chains have one or moremutations present in FW1, about 25% of the Vκ1-39/Jκ5-derived lightchains have one or more mutations present in CDR1, about 20% of theVκ1-39/Jκ5-derived light chains have one or more mutations present inFW2, about 10% of the Vκ1-39/Jκ5-derived light chains have one or moremutations present in CDR2, about 29% of the Vκ1-39/Jκ5-derived lightchains have one or more mutations present in FW3, and about 37% of theVκ1-39/Jκ5-derived light chains have one or more mutations present inCDR3.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 35% of the heavy chains have at least onemutation present in FW1; in one embodiment, at least 25% of the heavychains have one mutation present in FW1; in one embodiment, at least 9%of the heavy chains have two mutations present in FW1; in oneembodiment, at least 1% of the heavy chains have three mutations presentin FW1; in one embodiment, at least 1% of the heavy chains have morethan five mutations present in FW1.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 92% of the heavy chains have at least one or nomore than four mutations present in CDR1; in one embodiment, at least92% of the heavy chains have at least one, at least two, at least three,or at least four mutations present in CDR1; in one embodiment, at least26% of the heavy chains have one mutation present in CDR1; in oneembodiment, at least 44% of the heavy chains have two mutations presentin CDR1; in one embodiment, at least 19% of the heavy chains have threemutations present in CDR1; in one embodiment, at least 3% of the heavychains have four mutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 66% of the heavy chains have at least one or nomore than three mutations present in FW2; in one embodiment, at least66% of the heavy chains have at least one, at least two, or at leastthree mutations present in FW2; in one embodiment, at least 35% of theheavy chains have one mutation present in FW2; in one embodiment, atleast 23% of the heavy chains have two mutations present in FW2; in oneembodiment, at least 8% of the heavy chains have three mutations presentin FW2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 70% of the heavy chains have at least one or nomore than four mutations present in CDR2; in one embodiment, at least70% of the heavy chains have at least two, at least three, or at leastfour mutations present in CDR2; in one embodiment, at least 34% have onemutation present in CDR2; in one embodiment, at least 20% of the heavychains have two mutations present in CDR2; in one embodiment, at least12% of the heavy chains have three mutations present in CDR2; in oneembodiment, at least 5% of the heavy chains have four mutations presentin CDR2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 91% of the heavy chains have at least one or upto five or more mutations present in FW3; in one embodiment, at least91% of the heavy chains have at least two, at least three, at leastfour, or at least five or more mutations present in FW3; in oneembodiment, at least 19% of the heavy chains have one mutation presentin FW3; in one embodiment, at least 33% of the heavy chains have twomutations present in FW3; in one embodiment, at least 22% of the heavychains have three mutations present in FW3; in one embodiment, at least11% of the heavy chains have four mutations present in FW3; in oneembodiment, at least 7% of the heavy chains have five or more mutationspresent in FW3.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 63% of the heavy chains have at least one or nomore than two mutations present in CDR3; in one embodiment, at least 63%of the heavy chains have at one mutation present in CDR3; in oneembodiment, at least 54% of the heavy chains have one mutation presentin CDR3; in one embodiment, at least 9% of the heavy chains have twomutations present in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ1-39/Jκ5 rearrangement andat least 35% of the heavy chains have one or more mutations present inFW1, about 92% of the heavy chains have one or more mutations present inCDR1, about 66% of the heavy chains have one or more mutations presentin FW2, about 70% of the heavy chains have one or more mutations presentin CDR2, about 91% of the heavy chains have one or more mutationspresent in FW3, and about 63% of the heavy chains have one or moremutations present in CDR3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and the light chain gene has at least one or no more thantwo somatic hypermutations; in one embodiment, the light chain gene hasat least two, at least three, at least four or more somatichypermutations. In a specific embodiment, the mutations are present inone or more framework regions of the light chain. In a specificembodiment, the mutations are present in one or more CDR regions of thelight chain. In a specific embodiment, the mutations are present in oneor more framework regions and/or one or more CDR regions of the lightchain. In various embodiments, the framework regions are selected fromframework 1 (FW1), framework 2 (FW2), framework 3 (FW3), and/or acombination thereof.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 10% of the Vκ3-20/Jκ1-derived light chains haveat least one mutation present in FW1; in one embodiment, at least 10% ofthe light chains have one mutation in FW1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 53% of the Vκ3-20/Jκ1-derived light chains haveat least one or no more than two mutations present in CDR1; in oneembodiment, at least 27% of the light chains have one or more mutationsin CDR1; in one embodiment, about 54% of the light chains have one ortwo mutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 6% of the Vκ3-20/Jκ1-derived light chains haveat least one or no more than two mutations present in FW2; in oneembodiment, at least 6% of light chains have at least one mutationpresent in FW2; in one embodiment, at least 3% of the light chains haveone mutation present in FW2; in one embodiment, at least 3% of the lightchains have two mutations present in FW2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and at least about 3% of the Vκ3-20/Jκ1-derived lightchains have at least one mutation present in CDR2; in one embodiment, atleast 3% of the light chains have one mutation in CDR2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 17% or more of the Vκ3-20/Jκ1-derived lightchains have at least one or no more than two mutations present in FW3;in one embodiment, at least 20% of the light chain have one mutationpresent in FW3; in one embodiment, at least 17% of the light chains havetwo mutations present in FW3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and at least 43% of the Vκ3-20/Jκ1-derived light chainshave at least one mutation present in CDR3; in one embodiment, at least43% of the light chains have one mutation in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ3-20/Jκ1 rearrangement andabout 10% of the Vκ3-20/Jκ1-derived light chains have one or moremutations present in at least, about 53% of the Vκ3-20/Jκ1-derived lightchains have one or more mutations present in CDR1, about 6% of theVκ3-20/Jκ1-derived light chains have one or more mutations present inFW2, about 3% of the Vκ3-20/Jκ1-derived light chains have one or moremutations present in CDR2, about 37% of the Vκ3-20/Jκ1-derived lightchains have one or more mutations present in FW3, and about 43% of theVκ3-20/Jκ1-derived light chains have one or more mutations present inCDR3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 43% of the heavy chains have at least one or nomore than two mutations present in FW1; in one embodiment, at least 41%of the heavy chains have at least one mutation present in FW1; in oneembodiment, about 41% of the heavy chains have one mutation present inFW1; in one embodiment, about 2% of the heavy chains have two mutationspresent in FW1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 92% of the heavy chains have at least one or nomore than four mutations present in CDR1; in one embodiment, at least43% of heavy chains have at least one mutation present in CDR1; in oneembodiment, at least 25% of heavy chains have at least two mutationspresent in CDR1; in one embodiment, at least 15% of heavy chains have atleast 3 mutations present in CDR1; in one embodiment, at least 10% ofheavy chains have 4 or more mutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 46% of the heavy chains have at least one or nomore than three mutations present in FW2; in one embodiment, at least34% of heavy chains have at least one mutation present in FW2; in oneembodiment, at least 10% of heavy chains have two or more mutationspresent in FW2; in one embodiment, at least 2% of heavy chains havethree or more mutations present in FW2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 84% of the heavy chains have at least one or upto five or more than five mutations present in CDR2; in one embodiment,at least 39% of the heavy chains have one or more mutations present inCDR2; in one embodiment, at least 18% of the heavy chains have two ormore mutations present in CDR2; in one embodiment, at least 21% of theheavy chains have three or more mutations present in CDR2; in oneembodiment, at least 3% of the heavy chains have four or more mutationspresent in CDR2; in one embodiment, at least 2% of the heavy chains havefive or more mutations present in CDR2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 92% of the heavy chains have at least one or upto five or more than five mutations present in FW3; in one embodiment,at least 21% of the light chains have at least one mutation present inFW3; in one embodiment, at least 20% of heavy chains have at least twomutations present in FW3; in one embodiment, at least 13% of the heavychains have at least three mutations present in FW3; in one embodiment,at least 20% of the heavy chains have at least four mutations in FW3; inone embodiment, at least 18% of the heavy chains have at lest 5mutations in FW3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 7% of the heavy chains have at least onemutation present in CDR3; in one embodiment, about 7% of the heavychains have one mutation in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ3-20/Jκ1 rearrangement andabout 43% of the heavy chains have one or more mutations present in FW1,about 92% of the heavy chains have one or more mutations present inCDR1, about 46% of the heavy chains have one or more mutations presentin FW2, about 84% of the heavy chains have one or more mutations presentin CDR2, about 92% of the heavy chains have one or more mutationspresent in FW3, and about 7% of the heavy chains have one or moremutations present in CDR3.

In one aspect, a mouse that expresses an immunoglobulin light chain froma rearranged immunoglobulin light chain sequence is provided, whereinthe rearranged immunoglobulin light chain sequence is present in thegermline of the mouse, wherein the immunoglobulin light chain comprisesa human variable sequence. In one embodiment, the germline of the mousecomprises a rearranged immunoglobulin light chain sequence that isderived from the same V segment and the same J segment as allnon-surrogate light chain sequences present in every B cell of the mousethat comprises a rearranged light chain sequence.

In one embodiment, the germline of the mouse lacks a functionalunrearranged immunoglobulin light chain V gene segment. In oneembodiment, the germline of the mouse lacks a functional unrearrangedimmunoglobulin light chain J gene segment.

In one embodiment, the germline of the mouse comprises no more than one,no more than two, or no more than three rearranged (V/J) light chainsequences.

In one embodiment, the rearranged V/J sequence comprises a κ light chainsequence. In a specific embodiment, the κ light chain sequence is ahuman κ light chain sequence. In a specific embodiment, the κ lightchain sequence is selected from a human Vκ1-39/J sequence, a humanVκ3-20/J sequence, and a combination thereof. In a specific embodiment,the κ light chain sequence is a human Vκ1-39/Jκ5 sequence. In a specificembodiment, the κ light chain sequence is a human Vκ3-20/Jκ1 sequence.

In one embodiment, the mouse further comprises in its germline asequence selected from a mouse κ intronic enhancer 5′ with respect tothe rearranged immunoglobulin light chain sequence, a mouse κ 3′enhancer, and a combination thereof.

In one embodiment, the mouse comprises an unrearranged human V_(H) genesegment, an unrearranged human D_(H) gene segment, and an unrearrangedhuman J_(H) gene segment, wherein said V_(H), D_(H), and J_(H) genesegments are capable of rearranging to form an immunoglobulin heavychain variable gene sequence operably linked to a heavy chain constantgene sequence. In one embodiment, the mouse comprises a plurality ofhuman V_(H), D_(H), and J_(H) gene segments. In a specific embodiment,the human V_(H), D_(H), and J_(H) gene segments replace endogenous mouseV_(H), D_(H), and J_(H) gene segments at the endogenous mouseimmunoglobulin heavy chain locus. In a specific embodiment, the mousecomprises a replacement of all or substantially all functional mouseV_(H), D_(H), and J_(H) gene segments with all or substantially allfunctional human V_(H), D_(H), and J_(H) gene segments.

In one embodiment, the mouse expresses an immunoglobulin light chainthat comprises a mouse constant sequence. In one embodiment, the mouseexpresses an immunoglobulin light chain that comprises a human constantsequence.

In one embodiment, the mouse expresses an immunoglobulin heavy chainthat comprises a mouse sequence selected from a C_(H)1 sequence, a hingesequence, a C_(H)2 sequence, a C_(H)3 sequence, and a combinationthereof.

In one embodiment, the mouse expresses an immunoglobulin heavy chainthat comprises a human sequence selected from a C_(H)1 sequence, a hingesequence, a C_(H)2 sequence, a C_(H)3 sequence, and a combinationthereof.

In one embodiment, the rearranged immunoglobulin light chain sequence inthe germline of the mouse is at an endogenous mouse immunoglobulin lightchain locus. In a specific embodiment, the rearranged immunoglobulinlight chain sequence in the germline of the mouse replaces all orsubstantially all mouse light chain V and J sequences at the endogenousmouse immunoglobulin light chain locus.

In one aspect, a mouse is provided that comprises a B cell populationcharacterized by each B cell that comprises a non-surrogate light chainsequence, which sequence comprises a rearranged light chain gene that isgenerated from a single human V gene segment and a single human J genesegment, wherein the only light chain variable sequence in the germlineof the mouse is a rearranged sequence generated from the single human Vsegment and the single human J segment, and wherein each B cell thatcomprises the rearranged light chain gene further comprises a geneencoding a cognate human heavy chain variable domain, and wherein therearranged light chain gene comprises at least one, at least two, atleast three, or at least four somatic hypermutations.

In some embodiments, a mouse is provided whose mature B cell populationis characterized in that each mature B cell comprises a non-surrogatelight chain sequence on its surface, which sequence comprises arearranged light chain gene that is generated through rearrangement ofone of two human V_(L) gene segments and one of no more than five humanJ_(L) gene segments, wherein the only light chain variable sequence(V_(L)J_(L) sequence) in the germline of the mouse is a rearrangedsequence that is generated through rearrangement of one of the two humanV_(L) gene segments and one of the no more than five human J_(L) genesegments, and wherein each B cell that comprises the rearranged lightchain gene further comprises a gene encoding a cognate human heavy chainvariable domain, and wherein the rearranged light chain gene comprisesat least one, at least two, at least three, at least four, or five ormore somatic hypermutations. In some embodiments, a rearranged lightchain gene comprises one, two, three, four, or five somatichypermutations. In some embodiments, mice as described herein have beenimmunized with an antigen of interest, and, in some embodiments, amature B cell population is enriched with B cells that bind the antigenof interest.

In some embodiments, a mouse is provided whose mature B cell populationis characterized in that each mature B cell comprises a non-surrogatelight chain sequence on its surface, which sequence comprises arearranged light chain gene that is generated through rearrangement ofone of two human V_(L) gene segments and one of two or more (e.g., 2, 3,4, or 5) human J_(L) gene segments, wherein the V_(L) gene segmentsconsist essentially of two V_(L) gene segments that are not identicaland the V_(L) locus comprises two or more (e.g., 2, 3, 4, or 5) humanJ_(L) gene segments, and wherein each B cell that comprises therearranged light chain gene further comprises a gene encoding a cognatehuman heavy chain variable domain, and wherein the rearranged lightchain gene comprises at least one, at least two, at least three, atleast four, or five or more somatic hypermutations. In some embodiments,a rearranged light chain gene comprises one, two, three, four, or fivesomatic hypermutations. In some embodiments, mice as described hereinhave been immunized with an antigen of interest, and in someembodiments, a mature B cell population is enriched with B cells thatbind the antigen of interest.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a mouse as described herein is provided. In a specificembodiment, the cell is a mouse embryonic stem (ES) cell.

In one aspect, a tissue derived from a mouse as described herein isprovided. In one embodiment, the tissue is derived from spleen, lymphnode or bone marrow of a mouse as described herein.

In one aspect, a nucleus derived from a mouse as described herein isprovided. In one embodiment, the nucleus is from a diploid cell that isnot a B cell.

In one aspect, a mouse cell is provided that is isolated from a mouse asdescribed herein. In one embodiment, the cell is an ES cell. In oneembodiment, the cell is a lymphocyte. In one embodiment, the lymphocyteis a B cell. In one embodiment, the B cell expresses a chimeric heavychain comprising a variable domain derived from a human gene segment;and a light chain derived from a rearranged human Vκ1-39/J sequence,rearranged human Vκ3-20/J sequence, or a combination thereof; whereinthe heavy chain variable domain is fused to a mouse constant region andthe light chain variable domain is fused to a mouse or a human constantregion.

In one aspect, a hybridoma is provided, wherein the hybridoma is madewith a B cell of a mouse as described herein. In a specific embodiment,the B cell is from a mouse as described herein that has been immunizedwith an immunogen comprising an epitope of interest, and the B cellexpresses a binding protein that binds the epitope of interest, thebinding protein has a somatically mutated human V_(H) domain and a mouseC_(H), and has a human V_(L) domain derived from a rearranged humanVκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1 and a mouse C_(L).

In one aspect, a mouse embryo is provided, wherein the embryo comprisesa donor ES cell that is derived from a mouse as described herein.

In one aspect, a targeting vector is provided, comprising, from 5′ to 3′in transcriptional direction with reference to the sequences of the 5′and 3′ mouse homology arms of the vector, a 5′ mouse homology arm, ahuman or mouse immunoglobulin promoter, a human or mouse leadersequence, and a human V_(L) region selected from a rearranged humanVκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1, and a 3′ mouse homology arm.In one embodiment, the 5′ and 3′ homology arms target the vector to asequence 5′ with respect to an enhancer sequence that is present 5′ andproximal to the mouse Cκ gene. In one embodiment, the promoter is ahuman immunoglobulin variable region gene segment promoter. In aspecific embodiment, the promoter is a human Vκ3-15 promoter. In oneembodiment, the leader sequence is a mouse leader sequence. In aspecific embodiment, the mouse leader sequence is a mouse Vκ3-7 leadersequence.

In one aspect, a targeting vector is provided as described above, but inplace of the 5′ mouse homology arm the human or mouse promoter isflanked 5′ with a site-specific recombinase recognition site (SRRS), andin place of the 3′ mouse homology arm the human V_(L) region is flanked3′ with an SRRS.

In one aspect, a reverse chimeric antibody made by a mouse as describedherein, wherein the reverse chimeric antibody comprises a light chaincomprising a human V_(L) and a mouse C_(L), and a heavy chain comprisinga human V_(H) and a mouse C_(H).

In one aspect, a method for making an antibody is provided, comprisingexpressing in a single cell (a) a first V_(H) gene sequence of animmunized mouse as described herein fused with a human C_(H) genesequence; (b) a V_(L) gene sequence of an immunized mouse as describedherein fused with a human C_(L) gene sequence; and, (c) maintaining thecell under conditions sufficient to express a fully human antibody, andisolating the antibody. In one embodiment, the cell comprises a secondV_(H) gene sequence of a second immunized mouse as described hereinfused with a human C_(H) gene sequence, the first V_(H) gene sequenceencodes a V_(H) domain that recognizes a first epitope, and the secondV_(H) gene sequence encodes a V_(H) domain that recognizes a secondepitope, wherein the first epitope and the second epitope are notidentical.

In one aspect, a method for making an epitope-binding protein isprovided, comprising exposing a mouse as described herein with animmunogen that comprises an epitope of interest, maintaining the mouseunder conditions sufficient for the mouse to generate an immunoglobulinmolecule that specifically binds the epitope of interest, and isolatingthe immunoglobulin molecule that specifically binds the epitope ofinterest; wherein the epitope-binding protein comprises a heavy chainthat comprises a somatically mutated human V_(H) and a mouse C_(H),associated with a light chain comprising a mouse C_(L) and a human V_(L)derived from a rearranged human Vκ1-39Jκ5 or a rearranged humanVκ3-20Jκ1.

In one aspect, a cell that expresses an epitope-binding protein isprovided, wherein the cell comprises: (a) a human nucleotide sequenceencoding a human V_(L) domain that is derived from a rearranged humanVκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1, wherein the human nucleotidesequence is fused (directly or through a linker) to a humanimmunoglobulin light chain constant domain cDNA sequence (e.g., a humanκ constant domain DNA sequence); and, (b) a first human V_(H) nucleotidesequence encoding a human V_(H) domain derived from a first human V_(H)nucleotide sequence, wherein the first human V_(H) nucleotide sequenceis fused (directly or through a linker) to a human immunoglobulin heavychain constant domain cDNA sequence; wherein the epitope-binding proteinrecognizes a first epitope. In one embodiment, the epitope-bindingprotein binds the first epitope with a dissociation constant of lowerthan 10⁻⁶ M, lower than 10⁻⁸M, lower than 10⁻⁹ M, lower than 10⁻¹⁰ M,lower than 10⁻¹¹ M, or lower than 10⁻¹² M.

In one embodiment, the cell comprises a second human nucleotide sequenceencoding a second human V_(H) domain, wherein the second human sequenceis fused (directly or through a linker) to a human immunoglobulin heavychain constant domain cDNA sequence, and wherein the second human V_(H)domain does not specifically recognize the first epitope (e.g., displaysa dissociation constant of, e.g., 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, or higher),and wherein the epitope-binding protein recognizes the first epitope andthe second epitope, and wherein the first and the second immunoglobulinheavy chains each associate with an identical light chain of (a).

In one embodiment, the second V_(H) domain binds the second epitope witha dissociation constant that is lower than 10⁻⁶ M, lower than 10⁻⁷M,lower than 10⁻⁸ M, lower than 10⁻⁹ M, lower than 10⁻¹⁰ M, lower than10⁻¹¹ M, or lower than 10⁻¹² M.

In one embodiment, the epitope-binding protein comprises a firstimmunoglobulin heavy chain and a second immunoglobulin heavy chain, eachassociated with an identical light chain derived from a rearranged humanV_(L) region selected from a human Vκ1-39Jκ5 or a human Vκ3-20Jκ1,wherein the first immunoglobulin heavy chain binds a first epitope witha dissociation constant in the nanomolar to picomolar range, the secondimmunoglobulin heavy chain binds a second epitope with a dissociationconstant in the nanomolar to picomolar range, the first epitope and thesecond epitope are not identical, the first immunoglobulin heavy chaindoes not bind the second epitope or binds the second epitope with adissociation constant weaker than the micromolar range (e.g., themillimolar range), the second immunoglobulin heavy chain does not bindthe first epitope or binds the first epitope with a dissociationconstant weaker than the micromolar range (e.g., the millimolar range),and one or more of the V_(L), the V_(H) of the first immunoglobulinheavy chain, and the V_(H) of the second immunoglobulin heavy chain, aresomatically mutated.

In one embodiment, the first immunoglobulin heavy chain comprises aprotein A-binding residue, and the second immunoglobulin heavy chainlacks the protein A-binding residue.

In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and aretinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™cell).

In one aspect, a reverse chimeric antibody is provided, comprising ahuman V_(H) and a mouse heavy chain constant domain, a human V_(L) and amouse light chain constant domain, wherein the antibody is made by aprocess that comprises immunizing a mouse as described herein with animmunogen comprising an epitope, and the antibody specifically binds theepitope of the immunogen with which the mouse was immunized. In oneembodiment, the V_(L) domain is somatically mutated. In one embodimentthe V_(H) domain is somatically mutated. In one embodiment, both theV_(L) domain and the V_(H) domain are somatically mutated. In oneembodiment, the V_(L) is linked to a mouse Cκ domain.

In one aspect, a mouse is provided, comprising human V_(H) gene segmentsreplacing all or substantially all mouse V_(H) gene segments at theendogenous mouse heavy chain locus; no more than one or two rearrangedhuman light chain V_(L)/J_(L) sequences selected from a rearrangedVκ1-39/J and a rearranged Vκ3-20/J or a combination thereof, replacingall mouse light chain gene segments; wherein the human heavy chainvariable gene segments are linked to a mouse constant gene, and therearranged human light chain sequences are linked to a human or mouseconstant gene.

In some embodiments, a mouse is provided, comprising humanimmunoglobulin V_(H) gene segments replacing all or substantially allmouse immunoglobulin V_(H) gene segments at the endogenous mouseimmunoglobulin heavy chain locus; no more than two unrearranged humanimmunoglobulin V_(L) gene segments and two or more (e.g., 2, 3, 4 or 5)unrearranged human immunoglobulin J_(L) gene segments or five humanimmunoglobulin J_(L) gene segments, replacing all mouse immunoglobulinlight chain gene segments; wherein the human immunoglobulin V_(H) genesegments are linked to a mouse immunoglobulin constant gene, and theunrearranged human immunoglobulin V_(L) and J_(L) gene segments arelinked to a human or non-human immunoglobulin constant gene. In someembodiments, a non-human constant gene is a mouse immunoglobulinconstant gene. In some embodiments, a non-human immunoglobulin constantgene is a rat immunoglobulin constant gene.

In one aspect, a mouse ES cell comprising a replacement of all orsubstantially all mouse heavy chain variable gene segments with humanheavy chain variable gene segments, and no more than one or tworearranged human light chain V_(L)/J_(L) sequences, wherein the humanheavy chain variable gene segments are linked to a mouse immunoglobulinheavy chain constant gene, and the rearranged human light chainV_(L)/J_(L) sequences are linked to a mouse or human immunoglobulinlight chain constant gene. In a specific embodiment, the light chainconstant gene is a mouse constant gene.

In some embodiments, a mouse ES cell is provided, comprising areplacement of all or substantially all mouse immunoglobulin V_(H) genesegments with human immunoglobulin V_(H) gene segments and no more thantwo unrearranged human immunoglobulin V_(L) gene segments and two ormore (e.g., 2, 3, 4, or 5) unrearranged human immunoglobulin J_(L) genesegments, wherein the human immunoglobulin V_(H) gene segments arelinked to a mouse immunoglobulin heavy chain constant gene, and theunrearranged human immunoglobulin V_(L) and J_(L) gene segments arelinked to a non-human or human immunoglobulin light chain constant gene.In some certain embodiments, the non-human immunoglobulin light chainconstant gene is a mouse immunoglobulin constant gene. In some certainembodiments, the mouse comprises five unrearranged immunoglobulin J_(L)gene segments.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that at least one, and in some embodiments all, mouse V_(L)gene segments are replaced by one human V_(L) gene segment or no morethan two human V_(L) gene segments. In some embodiments, human V_(L)gene segments of a mouse are capable of rearranging to one of two ormore human J_(L) gene segments to encode an immunoglobulin V_(L) domainof an antibody. In some embodiments, human V_(L) gene segment(s) of alight chain locus of a mouse as described herein is/are operably linkedto two or more (e.g., two, three, four, or five) human J_(L) genesegments.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that it does not contain a nucleotide sequence beforerearrangement that encodes an endogenous V_(L) gene segment. In someembodiments, a mouse is provided, comprising a light chain locus whosestructure is different from that of the reference structure of FIG. 19in that it does not contain a nucleotide sequence before rearrangementthat encodes an endogenous J_(L) gene segment. In some embodiments, amouse is provided, comprising a light chain locus whose structure isdifferent from that of the reference structure of FIG. 19 in that itdoes not contain a nucleotide before rearrangement that encodesendogenous V_(L) and J_(L) gene segments.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that it does not contain a nucleotide sequence afterrearrangement that encodes an endogenous V_(L) gene segment. In someembodiments, a mouse is provided, comprising a light chain locus whosestructure is different from that of the reference structure of FIG. 19in that it does not contain a nucleotide sequence after rearrangementthat encodes an endogenous J_(L) gene segment. In some embodiments, amouse is provided, comprising a light chain locus whose structure isdifferent from that of the reference structure of FIG. 19 in that itdoes not contain a nucleotide sequence after rearrangement that encodesendogenous V_(L) and J_(L) gene segments.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that it contains no more than two human V_(L) gene segmentsand two or more (e.g., two, three, four, or five) human J_(L) genesegments before rearrangement. In some embodiments, a mouse is provided,comprising a light chain locus whose structure is different from that ofthe reference structure of FIG. 19 in that it contains no more than twohuman V_(L) gene segments and five human J_(L) gene segments beforerearrangement.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that it contains no more than two human V_(L) gene segmentsand five or less (e.g., 5, 4, 3, 2, or 1) human J_(L) gene segmentsafter rearrangement. In some embodiments, a mouse is provided,comprising a light chain locus whose structure is different from that ofthe reference structure of FIG. 19 in that it contains no more than twohuman V_(L) gene segments and one, two, three, four, or five human J_(L)gene segments after rearrangement.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that it contains one human V_(L) gene segment and five orless (e.g., 5, 4, 3, 2, or 1) human J_(L) gene segments afterrearrangement. In some embodiments, a mouse is provided, comprising alight chain locus whose structure is different from that of thereference structure of FIG. 19 in that it contains one human V_(L) genesegment and one, two, three, four, or five human J_(L) gene segmentsafter rearrangement.

In various embodiments, human V_(L) and J_(L) gene segments are human Vκand Jκ gene segments. In various embodiments, human Vκ segments areselected from a human Vκ1-39 gene segment and a human Vκ3-20 genesegment. In some embodiments, human Vκ segments are human Vκ1-39 andhuman Vκ3-20. In some embodiments, human Jκ segments are selected from aJκ1, Jκ2, Jκ3, Jκ4, Jκ5 gene segment, and a combination thereof. In someembodiments, human Jκ gene segments are human Jκ1, Jκ2, Jκ3, Jκ4, andJκ5.

In some embodiments, a mouse is provided, comprising a light chain locuswhose structure is different from that of the reference structure ofFIG. 19 in that it contains a structure that is substantially the sameas that of the structure of FIG. 1, FIG. 2, FIG. 3, or FIG. 9 beforerearrangement. In some embodiments, a mouse is provided, comprising alight chain locus whose structure is identical to the structure of FIG.1, FIG. 2, FIG. 3 or FIG. 9 before rearrangement.

In one aspect, an antigen-binding protein made by a mouse as describedherein is provided. In a specific embodiment, the antigen-bindingprotein comprises a human immunoglobulin heavy chain variable regionfused with a mouse constant region, and a human immunoglobulin lightchain variable region derived from a Vκ1-39 gene segment or a Vκ3-20gene segment, wherein the light chain constant region is a mouseconstant region.

In one aspect, a fully human antigen-binding protein made from animmunoglobulin variable region gene sequence from a mouse as describedherein is provided, wherein the antigen-binding protein comprises afully human heavy chain comprising a human variable region derived froma sequence of a mouse as described herein, and a fully human light chaincomprising a Vκ1-39 or a Vκ3-20. In one embodiment, the light chainvariable region comprises one to five somatic mutations. In oneembodiment, the light chain variable region is a cognate light chainvariable region that is paired in a B cell of the mouse with the heavychain variable region.

In one embodiment, the fully human antigen-binding protein comprises afirst heavy chain and a second heavy chain, wherein the first heavychain and the second heavy chain comprise non-identical variable regionsindependently derived from a mouse as described herein, and wherein eachof the first and second heavy chains express from a host cell associatedwith a human light chain derived from a Vκ1-39 gene segment or a Vκ3-20gene segment. In one embodiment, the first heavy chain comprises a firstheavy chain variable region that specifically binds a first epitope of afirst antigen, and the second heavy chain comprises a second heavy chainvariable region that specifically binds a second epitope of a secondantigen. In a specific embodiment, the first antigen and the secondantigen are different. In a specific embodiment, the first antigen andthe second antigen are the same, and the first epitope and the secondepitope are not identical; in a specific embodiment, binding of thefirst epitope by a first molecule of the binding protein does not blockbinding of the second epitope by a second molecule of the bindingprotein.

In one aspect, a fully human binding protein derived from a humanimmunoglobulin sequence of a mouse as described herein comprises a firstimmunoglobulin heavy chain and a second immunoglobulin heavy chain,wherein the first immunoglobulin heavy chain comprises a first variableregion that is not identical to a variable region of the secondimmunoglobulin heavy chain, and wherein the first immunoglobulin heavychain comprises a wild type protein A binding determinant, and thesecond heavy chain lacks a wild type protein A binding determinant. Inone embodiment, the first immunoglobulin heavy chain binds protein Aunder isolation conditions, and the second immunoglobulin heavy chaindoes not bind protein A or binds protein A at least 10-fold, ahundred-fold, or a thousand-fold weaker than the first immunoglobulinheavy chain binds protein A under isolation conditions. In a specificembodiment, the first and the second heavy chains are IgG1 isotypes,wherein the second heavy chain comprises a modification selected from95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein thefirst heavy chain lacks such modification.

In one aspect, a method for making a bispecific antigen-binding proteinis provided, comprising exposing a first mouse as described herein to afirst antigen of interest that comprises a first epitope, exposing asecond mouse as described herein to a second antigen of interest thatcomprises a second epitope, allowing the first and the second mouse toeach mount immune responses to the antigens of interest, identifying inthe first mouse a first human heavy chain variable region that binds thefirst epitope of the first antigen of interest, identifying in thesecond mouse a second human heavy chain variable region that binds thesecond epitope of the second antigen of interest, making a first fullyhuman heavy chain gene that encodes a first heavy chain that binds thefirst epitope of the first antigen of interest, making a second fullyhuman heavy chain gene that encodes a second heavy chain that binds thesecond epitope of the second antigen of interest, expressing the firstheavy chain and the second heavy chain in a cell that expresses a singlefully human light chain derived from a human Vκ1-39 or a human Vκ3-20gene segment to form a bispecific antigen-binding protein, and isolatingthe bispecific antigen-binding protein.

In one embodiment, the first antigen and the second antigen are notidentical.

In one embodiment, the first antigen and the second antigen areidentical, and the first epitope and the second epitope are notidentical. In one embodiment, binding of the first heavy chain variableregion to the first epitope does not block binding of the second heavychain variable region to the second epitope.

In one embodiment, the human light chain when paired with the firstheavy chain specifically binds the first epitope of the first antigenand when paired the second heavy chain specifically binds the secondepitope of the second antigen.

In one embodiment, the first antigen is selected from a soluble antigenand a cell surface antigen (e.g., a tumor antigen), and the secondantigen comprises a cell surface receptor. In a specific embodiment, thecell surface receptor is an immunoglobulin receptor. In a specificembodiment, the immunoglobulin receptor is an Fc receptor. In oneembodiment, the first antigen and the second antigen are the same cellsurface receptor, and binding of the first heavy chain to the firstepitope does not block binding of the second heavy chain to the secondepitope.

In one embodiment, the light chain variable domain of the light chaincomprises 2 to 5 somatic mutations. In one embodiment, the light chainvariable domain is a somatically mutated cognate light chain expressedin a B cell of the first or the second immunized mouse with either thefirst or the second heavy chain variable domain. In one embodiment, thelight chain of the cell comprises a germline sequence.

In one embodiment, the first fully human heavy chain bears an amino acidmodification that reduces its affinity to protein A, and the secondfully human heavy chain does not comprise a modification that reducesits affinity to protein A.

In one aspect, a method of preparing a bispecific antibody thatspecifically binds to a first and a second antigen is provided, whereinthe method comprises (a) identifying a first nucleic acid sequence thatencodes a first human heavy chain variable (V_(H)) domain that isspecific for the first antigen; (b) identifying a second nucleic acidsequence that encodes a second human heavy chain variable (V_(H)) domainthat is specific for the second antigen; (c) providing a third nucleicacid sequence that encodes a human light chain variable (V_(L)) regionwhich, when paired with the V_(H) region of (a) specifically binds thefirst antigen, and when paired with the V_(H) region of (b) specificallybinds to the second antigen; (d) culturing a host cell comprising thefirst, second, and third nucleic acid sequences to allow expression ofthe first and second human V_(H) regions and the human V_(L) region toform the bispecific antibody; and (d) recovering said bispecificantibody. In various aspects, the first and second antigens aredifferent from one another. In various aspects the first and secondnucleic acid sequences are isolated from an immunized mouse thatexpresses a human immunoglobulin V_(L) region from a rearrangedimmunoglobulin light chain sequence, wherein the rearrangedimmunoglobulin sequence is in the germline of the mouse.

In one embodiment, the human V_(L) region is derived from a rearrangedhuman light chain sequence comprising a human Vκ1-39 gene segment or ahuman Vκ3-20 gene segment. In a specific embodiment, the rearrangedhuman light chain sequence is a germline sequence (i.e., does notcomprise a somatic hypermutation within the V gene segment sequence).

In one embodiment, the third nucleic acid sequence is isolated from amouse that expresses a human immunoglobulin V_(L) region from arearranged immunoglobulin light chain sequence in the germline of themouse. In one embodiment, the rearranged immunoglobulin light chainsequence comprises a human Vκ1-39 or human Vκ3-20 gene segment. In aspecific embodiment, the rearranged immunoglobulin light chain sequencecomprises a human Vκ1-39 gene segment. In one embodiment, the humanimmunoglobulin V_(L) region is expressed from a modified endogenousimmunoglobulin light chain locus.

In one embodiment, the first and second antigens are present on onemolecule. In one embodiment, the first and second antigens are presenton different molecules. In various embodiments, the first or secondnucleic acid sequence comprises a modification that reduces the affinityof the encoded heavy chain to protein A.

In one embodiment, the first or second nucleic acid sequences comprise arearranged human heavy chain variable region sequence comprising a humanheavy chain gene segment selected from V_(H)1-2, V_(H)1-3, V_(H)1-8,V_(H)1-18, V_(H)1-24, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-33,V_(H)3-43, V_(H)3-48, V_(H)3-53, V_(H)3-64, V_(H)3-72, V_(H)3-73,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)5-51, and V_(H)6-1. Ina specific embodiment, the heavy chain gene segment is V_(H)2-5,V_(H)3-23 or V_(H)3-30.

In one aspect, a method of preparing a bispecific antibody thatspecifically binds to a first and a second antigen is provided, whereinthe method comprises (a) identifying a first nucleic acid sequence thatencodes a first human heavy chain variable (V_(H)) domain that isspecific for the first antigen; (b) identifying a second nucleic acidsequence that encodes a second human heavy chain variable (V_(H)) domainthat is specific for the second antigen; (c) providing a third nucleicacid sequence that encodes a human light chain variable (V_(L)) regionderived from a human Vκ1-39 or human Vκ3-20 gene segment which, whenpaired with the V_(H) region of (a) specifically binds the firstantigen, and when paired with the V_(H) region of (b) specifically bindsto the second antigen; (d) culturing a host cell comprising the first,second, and third nucleic acid sequences to allow expression of thefirst and second human V_(H) regions and the human V_(L) region to formthe bispecific antibody; and (d) recovering said bispecific antibody. Invarious aspects, the first and second antigens are different from oneanother. In various aspects, the first and second nucleic acid sequencesare isolated from an immunized mouse that expresses a humanimmunoglobulin V_(L) region from a rearranged immunoglobulin sequencethat is derived from a human Vκ1-39 or human Vκ3-20 gene segment,wherein the rearranged human Vκ1-39 or Vκ3-30 gene segment is in thegermline of the mouse.

In one embodiment, the third nucleic acid sequence is a germlinesequence (i.e., does not comprise a somatic hypermutation within the Vgene segment sequence). In one embodiment, the third nucleic acidsequence is isolated from the mouse that expresses a humanimmunoglobulin V_(L) region derived from a human Vκ1-39 or human Vκ3-20gene segment from a rearranged immunoglobulin light chain sequence inthe germline of the mouse. In a specific embodiment, the third nucleicacid sequence comprises two to five somatic hypermutations in acomplementary determining region (CDR) and/or a framework region (FWR).In one embodiment, the human immunoglobulin V_(L) region is expressedfrom a modified endogenous immunoglobulin light chain locus.

In one embodiment, the first and second antigens are present on onemolecule. In one embodiment, the first and second antigens are presenton different molecules. In one embodiment, the first or second nucleicacid sequence comprises a modification that reduces the affinity of theencoded heavy chain to protein A.

In one embodiment, the first or second nucleic acid sequences comprise arearranged human heavy chain variable region sequence comprising a humanheavy chain gene segment selected from V_(H)1-2, V_(H)1-3, V_(H)1-8,V_(H)1-18, V_(H)1-24, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-33,V_(H)3-43, V_(H)3-48, V_(H)3-53, V_(H)3-64, V_(H)3-72, V_(H)3-73,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)5-51, and V_(H)6-1. Ina specific embodiment, the heavy chain gene segment is V_(H)2-5,V_(H)3-23 or V_(H)3-30.

In one aspect, a method for making a bispecific antibody is provided,comprising exposing a mouse as described herein to an antigen ofinterest, allowing the mouse to mount an immune response to the antigenof interest, identifying a first human heavy chain variable region thatbinds a first epitope of the antigen of interest, identifying a secondhuman heavy chain variable region that binds a second epitope of theantigen of interest, making a first fully human heavy chain gene thatencodes the first heavy chain that binds the first epitope of theantigen of interest, making a second fully human heavy chain gene thatencodes a second heavy chain that binds the second epitope of theantigen of interest, expressing the first heavy chain and the secondheavy chain in a cell that expresses a single fully human light chainderived from a human Vκ1-39 or a human Vκ3-20 gene segment to form abispecific antibody, and isolating the bispecific antigen-bindingprotein.

In one embodiment, the first epitope and the second epitope are notidentical. In one embodiment, binding of the first heavy chain variableregion to the first epitope does not block binding of the second heavychain variable region to the second epitope. In one embodiment, thefirst and second heavy chains are capable of binding the first andsecond epitopes simultaneously.

In one embodiment, the bispecific antibody binds the first and secondepitopes simultaneously. In one embodiment, the bispecific antibodybinds the first epitope and second epitope independently.

In one embodiment, the binding response of the bispecific antibody tothe antigen is about 2-fold higher than the binding response of thefirst heavy chain variable region to the antigen. In one embodiment, thebinding response of the bispecific antibody to the antigen is about2-fold higher than the binding response of the second heavy chainvariable region to the antigen. In one embodiment, the binding responseof the bispecific antibody to the antigen is about the same as, or aboutequal to, the binding response of the first heavy chain variable regionand or the second heavy chain variable region to the antigen.

In one embodiment, the antigen is selected from a soluble antigen, acell surface antigen (e.g., a tumor antigen) and a cell surfacereceptor. In a specific embodiment, the cell surface receptor is animmunoglobulin receptor. In a specific embodiment, the immunoglobulinreceptor is an Fc receptor.

In one embodiment, the light chain variable domain of the light chaincomprises 2 to 5 somatic mutations. In one embodiment, the light chainvariable domain is a somatically mutated cognate light chain expressedin a B cell of the immunized mouse with either the first or the secondheavy chain variable domain.

In one embodiment, the first fully human heavy chain bears an amino acidmodification that reduces its affinity to protein A, and the secondfully human heavy chain does not comprise a modification that reducesits affinity to protein A.

In various embodiments, methods for making bispecific antibodies areenhanced by employing a common light chain to pair with each heavy chainvariable regions of the bispecific antibodies. In various embodiments,employing a common light chain as described herein reduces the number ofinappropriate species of immunoglobulins lacking bispecificity ascompared to employing original cognate light chains. In variousembodiments, the heavy chain variable regions of the bispecificantibodies are identified from monospecific antibodies comprising acommon light chain. In various embodiments, the heavy chain variableregions of the bispecific antibodies comprise human heavy chain variablegene segments that are rearranged in vivo within mouse B cells that havebeen previously engineered to express a limited human light chainrepertoire, or a single human light chain, cognate with human heavychains and, in response to exposure with an antigen of interest,generate a chimeric antibody repertoire containing a plurality of humanheavy chain variable regions that are cognate with one or one of twopossible human light chain variable regions, wherein the chimericantibodies are specific for the antigen of interest.

In various aspects, a method of preparing a bispecific antibody isprovided, the bispecific antibody comprising 1) a first polypeptide anda second polypeptide, wherein the first and second polypeptides eachinclude a multimerization domain (e.g., an immunoglobulin Fc domain)allowing the first and second polypeptides to form a dimer, and themultimerization domains promote stable interaction between first andsecond polypeptides, and wherein one of the multimerization domainsbears an amino acid modification that reduces its affinity to protein Aand the other multimerization domain lacks the modification, 2) abinding domain in each of the first and second polypeptide, each bindingdomain comprising a variable heavy chain and a variable light chain,wherein the variable light chain of the first polypeptide and thevariable light chain of the second polypeptide have a common amino acidsequence, which common sequence has an amino acid sequence identity toan original light chain of each of the polypeptides of at least 80%, ofat least 85%, preferably at least 90%, more preferably at least 95% andmost preferably 100% sequence identity. In various embodiments, thevariable light chain is derived from a human Vκ1-39 or a human Vκ3-20gene segment. In various embodiments, the variable light chain is arearranged human light chain sequence. In various embodiments, thevariable light chain is isolated from a mouse as described herein.

In various embodiments, the method comprises the steps of (i) culturinga host cell comprising a nucleic acid encoding the first polypeptide,the second polypeptide, and the common light chain, wherein the nucleicacid is expressed; and (ii) recovering the bispecific antibody from thehost cell culture; in one embodiment, the nucleic acid encoding thefirst polypeptide or the nucleic acid encoding the second polypeptide,bears an amino acid modification that reduces its affinity to protein A.In one embodiment, the nucleic acid encoding the first polypeptide, thesecond polypeptide, and the common light chain is present in a singlevector or in separate vectors. In one embodiment, the host cell is usedto make a bispecific antibody according to the preceding paragraph.

In one aspect, a method of preparing a bispecific antibody is provided,comprising (a) selecting a first nucleic acid encoding a first humanheavy chain variable region isolated from a mouse as described herein;(b) selecting a second nucleic acid encoding a second human heavy chainvariable region isolated from the same or separate mouse as describedherein; (c) providing a third nucleic acid encoding a human light chainvariable region isolated from a mouse as described herein or derivedfrom a rearranged human light chain variable region as described herein;(c) introducing into a host cell the first, second and third nucleicacids and culturing the host cell so that expression of the first,second and third nucleic acid occurs; and (d) recovering the bispecificantibody formed from the cell culture.

In one embodiment, the first and second human heavy chain variableregions are somatically mutated. In a specific embodiment, the first andsecond human heavy chain variable regions are independently derived froma rearranged human V_(H) gene segment selected from 1-2, 1-3, 1-8, 1-18,1-24, 1-46, 1-58, 1-69, 2-5, 2-26, 2-70, 3-7, 3-9, 3-11, 3-13, 3-15,3-16, 3-20, 3-21, 3-23, 3-30, 3-33, 3-43, 3-48, 3-53, 3-64, 3-72, 3-73,4-31, 4-34, 4-39, 4-59, 5-51, and a 6-1 human V_(H) gene segment. In oneembodiment, the first and second human heavy chain variable regions areindependently derived from a rearranged human V_(H) gene segmentselected from 2-5, 3-30 and 3-23. In one embodiment, the first humanheavy chain variable region is derived from a human V_(H)2-5 genesegment and the second human heavy chain variable region is derived froma human V_(H)3-30 gene segment. In one embodiment, the first human heavychain variable region is derived from a human V_(H)3-30 gene segment andthe second human heavy chain variable region is derived from a humanV_(H)3-23 gene segment. In one embodiment, the first human heavy chainvariable region is derived from a human V_(H)3-23 gene segment and thesecond human heavy chain variable region is derived from a humanV_(H)3-30 gene segment.

In one embodiment, the first or second nucleic acid is modified prior tostep (c), wherein the first or second nucleic acid is modified such thatit has a reduced affinity to protein A.

In one embodiment, the third nucleic acid is isolated from a mouse asdescribed herein. In one embodiment, the third nucleic acid comprises 2to 5 somatic mutations. In one embodiment, the third nucleic acidencodes a human light chain variable region derived from a human Vκ1-39gene segment. In one embodiment, the third nucleic acid encodes a humanlight chain variable region derived from a human Vκ3-20 gene segment.

In one embodiment, the third nucleic acid is derived from a rearrangedhuman light chain variable region. In one embodiment, the rearrangedhuman light chain variable region comprises a sequence derived from ahuman Vκ1-39 gene segment or a human Vκ3-20 gene segment. In oneembodiment, the rearranged human light chain variable region comprises agermline human Vκ1-39 sequence (i.e., does not comprise a somatichypermutation within the V gene segment sequence). In one embodiment,the rearranged human light chain variable region comprises a germlinehuman Vκ3-20 sequence.

In various embodiments, a method of preparing a bispecific antibody thatincorporates a first human heavy chain comprising a variable domainderived from a modified mouse that lacks a rearranged human light chainsequence in its germline is provided, wherein the first human heavychain is paired with a cognate human light chain that comprises arearranged human light chain variable region derived from a human Vκ1-39or a human Vκ3-20 gene segment. In various embodiments, a second humanheavy chain with a different specificity from the first human heavychain is identified from an immunized mouse as described herein. Nucleicacids encoding the two heavy chains and the common light chain areintroduced into a host cell as described in the preceding paragraphs sothat expression of all three chains occurs and the bispecific antibodyis recovered from the cell culture.

In one embodiment, the mouse is immunized with the same antigen used togenerate the first human heavy chain variable domain. In one embodiment,the mouse is immunized with a different antigen used to generate thefirst human heavy chain variable domain.

In one aspect, a method of selecting human heavy chains that can pairwith a single human light chain to make a bispecific antibody isprovided, including nucleic acids that encode the bispecific antibodyand a host cell comprising the nucleic acids.

In one aspect, a method of increasing the amount of a desired bispecificantibody in a cell culture over undesired products such as monospecificantibodies is provided, wherein one of the heavy chains of thebispecific antibody is modified to reduce its affinity to protein A.

In one aspect, an isolated host cell is provided, wherein the host cellcomprises (a) a first nucleic acid sequence encoding a first human heavychain variable region that binds a first antigen, wherein the firstnucleic acid sequence is isolated from a mouse immunized with the firstantigen that expresses a human immunoglobulin V_(L) region from arearranged immunoglobulin light chain sequence in the germline of themouse; (b) a second nucleic acid sequence encoding a second human heavychain variable region that binds a second antigen, wherein the secondnucleic acid sequence is isolated from a mouse immunized with the secondantigen that expresses a human immunoglobulin V_(L) region from arearranged immunoglobulin light chain sequence in the germline of themouse; (c) a third nucleic acid sequence encoding a human light chainvariable region which, when paired with the heavy chain variable regionof (a) specifically binds the first antigen, and when paired with theheavy chain variable region of (b) specifically binds to the secondantigen.

In various aspects, the first and second antigens are different from oneanother. In various aspects, the expression of the first, second andthird nucleic acid sequences leads to the formation of a bispecificantibody that specifically binds to the first and second antigens.

In one embodiment, the human V_(L) region is derived from a rearrangedhuman light chain sequence comprising a human Vκ1-39 gene segment or ahuman Vκ3-20 gene segment. In a specific embodiment, the rearrangedhuman light chain sequence is a germline sequence (i.e., does notcomprise a somatic hypermutation within the variable domain). In oneembodiment, the third nucleic acid sequence is isolated from a mousethat expresses a human immunoglobulin V_(L) region from a rearrangedimmunoglobulin light chain sequence, wherein the rearranged human lightchain sequence is present in the germline of the mouse. In oneembodiment, the rearranged immunoglobulin light chain sequence comprisesa human Vκ1-39 gene segment or a human Vκ3-20 gene segment. In aspecific embodiment, the human Vκ1-39 gene segment or human Vκ3-20 genesegment comprises at least one somatic hypermutation in a complementarydetermining region (CDR) or framework region (FWR). In a specificembodiment, the first, second and third nucleic acid sequences areisolated from a mouse that expresses a human immunoglobulin V_(L) regionderived from a human Vκ1-39 or human Vκ3-20 gene segment from arearranged immunoglobulin light chain sequence, wherein the rearrangedimmunoglobulin light chain sequence is present in the germline of themouse.

In various embodiments, the mouse does not contain an endogenous lightchain variable region gene segment that is capable of rearranging toform an immunoglobulin light chain.

In one embodiment, the human immunoglobulin V_(L) region is expressedfrom a modified endogenous immunoglobulin light chain locus. In oneembodiment, the first and second antigens are present on one molecule.In one embodiment, the first and second antigens are present ondifferent molecules. In one embodiment, the first or second nucleic acidsequence comprises a modification that reduces the affinity of theencoded heavy chain to protein A.

In one embodiment, the first or second nucleic acid sequences comprise arearranged human heavy chain variable region sequence comprising a humanheavy chain gene segment selected from V_(H)1-2, V_(H)1-3, V_(H)1-8,V_(H)1-18, V_(H)1-24, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-33,V_(H)3-43, V_(H)3-48, V_(H)3-53, V_(H)3-64, V_(H)3-72, V_(H)3-73,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)5-51, and V_(H)6-1. Ina specific embodiment, the heavy chain gene segment is V_(H)2-5,V_(H)3-23 or V_(H)3-30.

In one aspect, an antibody or a bispecific antibody comprising a humanheavy chain variable domain made in accordance with the invention isprovided. In another aspect, use of a mouse as described herein to makea fully human antibody or a fully human bispecific antibody is provided.

In one aspect, a genetically modified mouse, embryo, or cell describedherein comprises a κ light chain locus that retains endogenousregulatory or control elements, e.g., a mouse κ intronic enhancer, amouse κ 3′ enhancer, or both an intronic enhancer and a 3′ enhancer,wherein the regulatory or control elements facilitate somatic mutationand affinity maturation of an expressed sequence of the κ light chainlocus.

In one aspect, a mouse is provided that comprises a B cell populationcharacterized by having immunoglobulin light chains derived from no morethan one, or no more than two, rearranged or unrearranged immunoglobulinlight chain V and J gene segments, wherein the mouse exhibits a κ:λlight chain ratio that is about the same as a mouse that comprises awild type complement of immunoglobulin light chain V and J genesegments.

In one embodiment, the immunoglobulin light chains are derived from nomore than one, or no more than two, rearranged immunoglobulin lightchain V and J gene segments. In a specific embodiment, the light chainsare derived from no more than one rearranged immunoglobulin light chainV and J gene segments. In one embodiment, the immunoglobulin lightchains are generated from one of two unrearranged immunoglobulin V_(L)gene segments and one of 1, 2, 3, 4, or 5 immunoglobulin J_(L) genesegments. In one embodiment, the immunoglobulin light chains aregenerated from one of two unrearranged immunoglobulin V_(L) genesegments and one immunoglobulin J_(L) gene segment.

In one aspect, a mouse as described herein is provided that expresses animmunoglobulin light chain derived from no more than one, or no morethan two, human Vκ/Jκ sequences, wherein the mouse comprises areplacement of all or substantially all endogenous mouse heavy chainvariable region gene segments with one or more human heavy chainvariable region gene segments, and the mouse exhibits a ratio of (a)CD19⁺ B cells that express an immunoglobulin having a λ light chain, to(b) CD19⁺ B cells that express an immunoglobulin having a κ light chain,of about 1 to about 20.

In one embodiment, the mouse expresses a single κ light chain derivedfrom a human Vκ1-39Jκ5 sequence, and the ratio of CD19⁺ B cells thatexpress an immunoglobulin having a λ light chain to CD19⁺ B cells thatexpress an immunoglobulin having a κ light chain is about 1 to about 20;in one embodiment, the ratio is about 1 to at least about 66; in aspecific embodiment, the ratio is about 1 to 66.

In one embodiment, the mouse expresses a single κ light chain derivedfrom a human Vκ3-20Jκ5 sequence, and the ratio of CD19⁺ B cells thatexpress an immunoglobulin having a λ light chain to CD19⁺ B cells thatexpress an immunoglobulin having a κ light chain is about 1 to about 20;in one embodiment, the ratio is about 1 to about 21. In specificembodiments, the ratio is 1 to 20, or 1 to 21.

In some embodiments, the present invention provides a mouse thatexpresses an immunoglobulin light chain whose sequence is identical tothat achieved by rearrangement of one of two human Vκ gene segments with1, 2, 3, 4, or 5 human Jκ gene segments.

In some embodiments, a mouse is provided that expresses animmunoglobulin light chain generated from a rearrangement of one of twohuman Vκ gene segments and one of 1, 2, 3, 4, or 5 human Jκ genesegments, wherein the mouse comprises a replacement of all orsubstantially all endogenous immunoglobulin V_(H) gene segments with oneor more human immunoglobulin V_(H), one or more D_(H), and one or moreJ_(H) gene segments, and the mouse exhibits a ratio of (a) B cells inthe bone marrow that express an immunoglobulin having a λ light chain,to (b) B cells in the bone marrow that express an immunoglobulin havinga κ light chain, of about 1 to about 15. In some embodiments, therearrangement includes a human Vκ1-39 gene segment. In some embodiments,the rearrangement includes a human Vκ3-20 gene segment. In someembodiments, the replacement of the endogenous immunoglobulin V_(H) genesegments is at an endogenous immunoglobulin V_(H) locus. In someembodiments, the two human Vκ gene segments is at an endogenousimmunoglobulin Vκ locus, and, in some embodiments, the two human Vκ genesegments replace all or substantially all mouse immunoglobulin Vκ genesegments. In some embodiments, the two human Vκ gene segments are at anendogenous immunoglobulin Vκ locus, and, in some embodiments, the twohuman Vκ gene segments replace all or substantially all mouseimmunoglobulin Vκ and Jκ gene segments. In various embodiments, the twohuman Vκ gene segments are operably linked to two or more (e.g., 2, 3,4, 5) human Jκ gene segments.

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and the ratio of immature B cells in the bonemarrow that express an immunoglobulin having a λ light chain to immatureB cells that express an immunoglobulin having a κ light chain is about 1to about 13.

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and the ratio of mature B cells in the bonemarrow that express an immunoglobulin having a λ light chain to immatureB cells that express an immunoglobulin having a κ light chain is about 1to about 7.

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a pro B cell population in the bonemarrow within in the range of about 2.5×10⁴ to about 1.5×10⁵ cells,inclusive, for example about 2.5×10⁴, 3.0×10⁴, 3.5×10⁴, 4.0×10⁴,4.5×10⁴, 5.0×10⁴, 5.5×10⁴, 6.0×10⁴, 6.5×10⁴, 7.0×10⁴, 7.5×10⁴, 8.0×10⁴,8.5×10⁴, 9.0×10⁴, 9.5×10⁴, 1.0×10⁵, or 1.5×10⁵ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 2.88×10⁴ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 6.42×10⁴ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 9.16×10⁴ cells; in someembodiments, a mouse of the present invention comprises a pro B cellpopulation in the bone marrow of about 1.19×10⁵ cells. Exemplary pro Bcells in the bone marrow of genetically modified mice as describedherein are characterized by expression of CD19, CD43, c-kit and/or acombination thereof (e.g., CD19⁺, CD43⁺, c-kit⁺).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a pre B cell population in the bonemarrow within in the range of about 1×10⁶ to about 2×10⁶ cells,inclusive, for example, about 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶,1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or 2.0×10⁶ cells;in some embodiments, a mouse of the present invention comprises a pre Bcell population in the bone marrow of about 1.25×10⁶ cells; in someembodiments, a mouse of the present invention comprises a pre B cellpopulation in the bone marrow of about 1.46×10⁶ cells; in someembodiments, a mouse of the present invention comprises a pre B cellpopulation in the bone marrow of about 1.64×10⁶ cells; in someembodiments, a mouse of the present invention comprises a pre B cellpopulation in the bone marrow of about 2.03×10⁶ cells. Exemplary pre Bcells in the bone marrow of genetically modified mice as describedherein are characterized by expression of CD19, CD43, c-kit and/or acombination thereof (e.g., CD19⁺, CD43⁻, c-kit⁻).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has an immature B cell population in thebone marrow within the range of about 5×10⁵ to about 7×10⁵ cells,inclusive, for example, about 5.0×105, 5.1×105, 5.2×10⁵, 5.3×10⁵,5.4×10⁵, 5.5×10⁵, 5.6×10⁵, 5.7×10⁵, 5.8×10⁵, 5.9×10⁵, 6.0×10⁵, 6.1×10⁵,6.2×10⁵, 6.3×10⁵, 6.4×10⁵, 6.5×10⁵, 6.6×10⁵, 6.7×10⁵, 6.8×10⁵, 6.9×10⁵,or 7.0×10⁵ cells; in some embodiments, a mouse of the present inventioncomprises an immature B cell population in the bone marrow of about5.33×10⁵ cells; in some embodiments, a mouse of the present inventioncomprises an immature B cell population in the bone marrow of about5.80×10⁵ cells; in some embodiments, a mouse of the present inventioncomprises an immature B cell population in the bone marrow of about5.92×10⁵ cells; in some embodiments, the mouse comprises an immature Bcell population in the bone marrow of about 6.67×10⁵ cells. Exemplaryimmature B cells in the bone marrow of genetically modified mice asdescribed herein are characterized by expression of IgM, B220 and/or acombination thereof (e.g., IgM⁺, B220^(int)).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a mature B cell population in thebone marrow within the range of about 3×10⁴ to about 1.5×10⁵ cells,inclusive, for example about 3.0×10⁴, 3.5×10⁴, 4.0×10⁴, 4.5×10⁴,5.0×10⁴, 5.5×10⁴, 6.0×10⁴, 6.5×10⁴, 7.0×10⁴, 7.5×10⁴, 8.0×10⁴, 8.5×10⁴,9.0×10⁴, 9.5×10⁴, 1.0×10⁵, or 1.5×10⁵ cells; in some embodiments, amouse of the present invention comprises a mature B cell population inthe bone marrow of about 3.11×10⁴ cells; in some embodiments, a mouse ofthe present invention comprise a mature B cell population in the bonemarrow of about 1.09×10⁵ cells; in some embodiments, a mouse of thepresent invention comprises a mature B cell population in the bonemarrow of about 1.16×10⁵ cells; in some embodiments, a mouse of thepresent invention comprises a mature B cell population in the bonemarrow of about 1.44×10⁵ cells. Exemplary mature B cells in the bonemarrow of genetically modified mice as described herein arecharacterized by expression of IgM, B220 and/or a combination thereof(e.g., IgM⁺, B220^(hi)).

In some embodiments, a mouse of the present invention expresses a lightchain generated through a rearrangement of a human Vκ1-39 gene segmentor a human Vκ3-20 gene segment and one of two or more (e.g., 2, 3, 4, or5) human Jκ gene segments, and has a total B cell population in the bonemarrow within the range of about 1×10⁶ to about 3×10⁶ cells, inclusive,for example about 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶,1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶,2.4×10⁶, 2.5×10⁶, 2.6×10⁶, 2.7×10⁶, 2.8×10⁶, 2.9×10⁶ or 2.0×10⁶ cells;in some embodiments, a mouse of the present invention comprises a totalB cell population in the bone marrow of about 1.59×10⁶ cells; in someembodiments, a mouse of the present invention comprises a total B cellpopulation in the bone marrow of about 1.75×10⁶ cells; in someembodiments, a mouse of the present invention comprises a total B cellpopulation in the bone marrow of about 2.13×10⁶ cells; in someembodiments, a mouse of the present invention comprises a total B cellpopulation in the bone marrow of about 2.55×10⁶ cells. An exemplarytotal B cells in the bone marrow of genetically modified mice asdescribed herein are characterized by expression CD19, CD20 and/or acombination thereof (e.g., CD19⁺).

In one aspect, a genetically modified mouse is provided that expresses asingle rearranged κ light chain, wherein the mouse comprises afunctional λ light chain locus, and wherein the mouse expresses a B cellpopulation that comprises Igκ⁺ cells that express a κ light chainderived from the same single rearranged κ light chain. In oneembodiment, the percent of Igκ⁺Igλ⁺ B cells in the mouse is about thesame as in a wild type mouse. In a specific embodiment, the percent ofIgκ⁺Igλ⁺ B cells in the mouse is about 2 to about 6 percent. In aspecific embodiment, the percent of Igκ⁺Igλ⁺ B cells in a mouse whereinthe single rearranged κ light chain is derived from a Vκ1-39Jκ5 sequenceis about 2 to about 3; in a specific embodiment, the percent is about2.6. In a specific embodiment, the percent of Igκ⁺Igλ⁺ B cells in amouse wherein the single rearranged κ light chain is derived from aVκ3-20Jκ1 sequence is about 4 to about 8; in a specific embodiment, thepercent is about 6.

In some embodiments, a genetically modified mouse is provided thatexpresses an immunoglobulin light chain comprising a rearranged humanimmunoglobulin Vκ/Jκ sequence, wherein the mouse comprises a functionalimmunoglobulin λ light chain locus, and wherein the mouse comprises asplenic B cell population that comprises a ratio of Igλ⁺ B cells to Igκ⁺B cells that is about 1 to about 8; in some embodiments, about 1 toabout 5. In some embodiments, the rearranged human immunoglobulin Vκ/Jκsequence is generated through a rearrangement of one of two humanimmunoglobulin Vκ gene segments and one of 1, 2, 3, 4, or 5 humanimmunoglobulin Jκ gene segments. In some embodiments, the rearrangedhuman immunoglobulin Vκ/Jκ sequence is generated through a rearrangementof a human immunoglobulin Vκ1-39 gene segment and a human immunoglobulinJκ gene segment selected from Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combinationthereof. In some embodiments, the rearranged human immunoglobulin Vκ/Jκsequence is generated through a rearrangement of a human immunoglobulinVκ3-20 gene segment and a human immunoglobulin Jκ gene segment selectedfrom Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combination thereof.

In some embodiments, a mouse of the present invention comprises a CD19⁺splenic B cell population within the range of about 2×10⁶ to about 7×10⁶cells, inclusive, for example about 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or 7.0×10⁶ cells;in some embodiments, a mouse of the present invention comprises a CD19⁺splenic B cell population of about 2.74×10⁶ cells; some embodiments, amouse of the present invention comprises a CD19⁺ splenic B cellpopulation of about 4.30×10⁶ cells; in some embodiments, a mouse of thepresent invention comprises a CD19⁺ splenic B cell population of about5.53×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a CD19⁺ splenic B cell population of about 6.18×10⁶ cells.

In some embodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population within the range of about1×10⁶ to about 4×10⁶ cells, inclusive, for example about 1.0×10⁶,1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, 4.0×10⁶ cells; in someembodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 1.30×10⁶; in someembodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 2.13×10⁶ cells; insome embodiments, a mouse of the present invention comprises CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 3.15×10⁶ cells; insome embodiments, a mouse of the present invention comprises a CD19⁺,IgD^(hi), IgM^(lo) splenic B cell population of about 3.93×10⁶ cells.

In some embodiment, a mouse of the present invention comprises a CD19⁺,IgD^(lo), IgM^(hi) splenic B cell population within the range of about9×10⁵ to about 2×10⁶ cells, inclusive, for example about 9.0×10⁵,9.25×10⁵, 9.5×10⁵, 9.75×10⁵, 1.0×10⁶, 1.25×10⁶, 1.50×10⁶, 1.75×10⁶,2.0×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about9.52×10⁵; in some embodiments, a mouse of the present inventioncomprises a CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about1.23×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about1.40×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population of about1.42×10⁶ cells.

In some embodiments, a genetically modified mouse is provided, whereinthe mouse comprises an immunoglobulin κ light chain locus that comprisestwo unrearranged human immunoglobulin Vκ gene segments and two or more(e.g., 2, 3, 4, or 5) unrearranged human Jκ gene segments, and whereinthe mouse comprises a peripheral splenic B cell population comprisingtransitional (e.g., T1, T2 and T3) B cell populations that are about thesame as a mouse that comprises a wild type complement of immunoglobulinκ light chain V and J gene segments. Exemplary transitional B cellpopulations (e.g., T1, T2 and T3) in the spleen of a geneticallymodified mouse as described herein are characterized by expression ofIgM, CD23, CD93, B220 and/or a combination thereof.

In some embodiments, a mouse of the present invention comprises a T1 Bcell population in the spleen (e.g., CD93⁺, B220⁺, IgM^(hi), CD23⁻)within the range of about 2×10⁶ to about 7×10⁶ cells, inclusive, forexample about 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, 4.0×10⁶, 4.5×10⁶,5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or 7.0×10⁶ cells; in someembodiments, a mouse of the present invention comprises a T1 B cellpopulation in the spleen of about 2.16×10⁶ cells; in some embodiments, amouse of the present invention comprises a T1 B cell population in thespleen of about 3.63×10⁶ cells; in some embodiments, a mouse of thepresent invention comprises a T1 B cell population in the spleen ofabout 3.91×10⁶; in some embodiments, a mouse of the present inventioncomprises a T1 B cell population in the spleen of about 6.83×10⁶ cells.

In some embodiments, a mouse of the present invention comprises a T2 Bcell population in the spleen (e.g., CD93⁺, B220⁺, IgM^(hi), CD23⁺)within the range of about 1×10⁶ to about 7×10⁶ cells, inclusive, forexample about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or 7.0×10⁶ cells;in some embodiments, a mouse of the present invention mouse comprises aT2 B cell population in the spleen of about 1.30×10⁶ cells; in someembodiments, a mouse of the present invention comprises a T2 B cellpopulation in the spleen of about 2.46×10⁶ cells; in some embodiments, amouse of the present invention comprises a T2 B cell population in thespleen of about 3.24×10⁶; in some embodiments, a mouse of the presentinvention comprises a T2 B cell population in the spleen of about6.52×10⁶ cells.

In some embodiments, a mouse of the present invention comprises a T3 Bcell population in the spleen (e.g., CD93⁺, B220⁺, IgM^(lo), CD23⁺)within the range of about 1×10⁶ to about 4×10⁶ cells, inclusive, forexample about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, or4.0×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a T3 B cell population in the spleen of about 1.08×10⁶ cells;in some embodiments, a mouse of the present invention comprises a T3 Bcell population in the spleen of about 1.35×10⁶ cells; in someembodiments, a mouse of the present invention comprises a T3 B cellpopulation in the spleen of about 3.37×10⁶; in some embodiments, a mouseof the present invention comprises a T1 B cell population in the spleenof about 3.63×10⁶ cells.

In some embodiments, a genetically modified mouse is provided, whereinthe mouse comprises an immunoglobulin κ light chain locus that comprisestwo unrearranged human immunoglobulin Vκ gene segments and 1, 2, 3, 4,or 5 unrearranged human immunoglobulin Jκ gene segments, and wherein themouse comprises a peripheral splenic B cell population comprisingmarginal zone and marginal zone precursor B cell populations that areabout the same as a mouse that comprises a wild type complement ofimmunoglobulin Vκ and Jκ gene segments. Exemplary marginal zone B cellpopulations in the spleen of a genetically modified mouse as describedherein are characterized by expression of IgM, CD21/35, CD23, CD93, B220and/or a combination thereof.

In some embodiments, a mouse of the present invention comprises marginalzone B cell population in the spleen (e.g., CD93⁻, B220⁺, IgM^(hi),CD21/35^(hi), CD23⁻) within the range of about 1×10⁶ to about 3×10⁶cells, inclusive, for example, about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶,or 3.0×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about1.47×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about1.49×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about2.26×10⁶ cells; in some embodiments, a mouse of the present inventioncomprises a marginal zone B cell population in the spleen of about2.33×10⁶ cells.

In some embodiments, a genetically modified mouse is provided, whereinthe mouse comprises an immunoglobulin κ light chain locus that comprisestwo unrearranged human immunoglobulin Vκ gene segments and 1, 2, 3, 4,or 5 unrearranged human immunoglobulin Jκ gene segments, and wherein themouse comprises a peripheral splenic B cell population comprisingfollicular (e.g., FO-I and FO-II) B cell population(s) that are aboutthe same as a mouse that comprises a wild type complement ofimmunoglobulin Vκ and Jκ gene segments. Exemplary follicular B cellpopulations (e.g., FO-I and FO-II) in the spleen of a geneticallymodified mouse as described herein are characterized by expression ofIgM, IgD, CD21/35, CD93, B220 and/or a combination thereof.

In some embodiments, a mouse of the present invention comprises afollicular type 1 B cell population in the spleen (e.g., CD93⁻, B220⁺,CD21/35^(int), IgM^(lo), IgD^(hi)) within the range of about 3×10⁶ toabout 1.5×10⁷ cells, inclusive, for example about 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, 7.0×10⁶, 7.5×10⁶,8.0×10⁶, 8.5×10⁶, 9.0×10⁶, 9.5×10⁶, 1.0×10⁷, or 1.5×10⁷ cells; in someembodiments, a mouse of the present invention comprises a folliculartype 1 B cell population in the spleen of about 3.57×10⁶ cells; in someembodiments, a mouse of the present invention comprises a folliculartype 1 B cell population in the spleen of about 6.31×10⁶ cells; in someembodiments, a mouse of the present invention comprises a folliculartype 1 B cell population in the spleen of about 9.42×10⁶ cells; in someembodiments, a mouse of the present invention comprise a follicular type1 B cell population in the spleen of about 1.14×10⁷ cells.

In some embodiments, a mouse of the present invention comprises afollicular type 2 B cell population in the spleen (e.g., CD93⁻, B220⁺,CD21/35^(int), IgM^(int), IgD^(hi)) within the range of about 1×10⁶ toabout 2×10⁶ cells, inclusive, for example, 1.0×10⁶, 1.25×10⁶, 1.5×10⁶,1.75×10⁶, or 2.0×10⁶ cells; in some embodiments, a mouse of the presentinvention comprises a follicular type 2 B cell population in the spleenof about 1.14×10⁶ cells; in some embodiments, a mouse of the presentinvention comprises a follicular type 2 B cell population in the spleenof about 1.45×10⁶ cells; in some embodiments, a mouse of the presentinvention comprises a follicular type 2 B cell population in the spleenof about 1.80×10⁶; in some embodiments, a mouse of the present inventioncomprise a follicular type 2 B cell population in the spleen of about2.06×10⁶ cells.

In one aspect, a genetically modified mouse is provided, wherein themouse expresses a single rearranged κ light chain derived from a humanVκ and Jκ gene segment, wherein the mouse expresses a B cell populationthat comprises a single κ light chain derived from the single rearrangedκ light chain sequence, wherein the genetically modified mouse has notbeen rendered resistant to somatic hypermutations. In one embodiment, atleast 90% of the κ light chains expressed on a B cell of the mouseexhibit from at least one to about five somatic hypermutations.

In one aspect, a genetically modified mouse is provided that is modifiedto express a single κ light chain derived from no more than one, or nomore than two, rearranged κ light chain sequences, wherein the mouseexhibits a κ light chain usage that is about two-fold or more, at leastabout three-fold or more, or at least about four-fold or more greaterthan the κ light chain usage exhibited by a wild type mouse, or greaterthan the κ light chain usage exhibited by a mouse of the same strainthat comprises a wild type repertoire of κ light chain gene segments. Ina specific embodiment, the mouse expresses the single κ light chain fromno more than one rearranged κ light chain sequence. In a more specificembodiment, the rearranged κ light chain sequence is selected from aVκ1-39Jκ5 and Vκ3-20Jκ1 sequence. In one embodiment, the rearranged κlight chain sequence is a Vκ1-39Jκ5 sequence. In one embodiment, therearranged κ light chain sequence is a Vκ3-20Jκ1 sequence.

In one aspect, a genetically modified mouse is provided that expresses asingle κ light chain derived from no more than one, or no more than two,rearranged κ light chain sequences, wherein the mouse exhibits a κ lightchain usage that is about 100-fold or more, at least about 200-fold ormore, at least about 300-fold or more, at least about 400-fold or more,at least about 500-fold or more, at least about 600-fold or more, atleast about 700-fold or more, at least about 800-fold or more, at leastabout 900-fold or more, at least about 1000-fold or more greater thanthe same κ light chain usage exhibited by a mouse bearing a complete orsubstantially complete human κ light chain locus. In a specificembodiment, the mouse bearing a complete or substantially complete humanκ light chain locus lacks a functional unrearranged mouse κ light chainsequence. In a specific embodiment, the mouse expresses the single κlight chain from no more than one rearranged κ light chain sequence. Inone embodiment, the mouse comprises one copy of a rearranged κ lightchain sequence (e.g., a heterozygote). In one embodiment, the mousecomprises two copies of a rearranged κ light chain sequence (e.g., ahomozygote). In a more specific embodiment, the rearranged κ light chainsequence is selected from a Vκ1-39Jκ5 and Vκ3-20Jκ1 sequence. In oneembodiment, the rearranged κ light chain sequence is a Vκ1-39Jκ5sequence. In one embodiment, the rearranged κ light chain sequence is aVκ3-20Jκ1 sequence.

In one aspect, a genetically modified mouse is provided that expresses asingle light chain derived from no more than one, or no more than two,rearranged light chain sequences, wherein the light chain in thegenetically modified mouse exhibits a level of expression that is atleast 10-fold to about 1,000-fold, 100-fold to about 1,000-fold,200-fold to about 1,000-fold, 300-fold to about 1,000-fold, 400-fold toabout 1,000-fold, 500-fold to about 1,000-fold, 600-fold to about1,000-fold, 700-fold to about 1,000-fold, 800-fold to about 1,000-fold,or 900-fold to about 1,000-fold higher than expression of the samerearranged light chain exhibited by a mouse bearing a complete orsubstantially complete light chain locus. In one embodiment, the lightchain comprises a human sequence. In a specific embodiment, the humansequence is a κ sequence. In one embodiment, the human sequence is a λsequence. In one embodiment, the light chain is a fully human lightchain.

In one embodiment, the level of expression is characterized byquantitating mRNA of transcribed light chain sequence, and comparing itto transcribed light chain sequence of a mouse bearing a complete orsubstantially complete light chain locus.

In one aspect, a genetically modified mouse is provided that expresses asingle κ light chain derived from no more than one, or no more than two,rearranged κ light chain sequences, wherein the mouse, upon immunizationwith antigen, exhibits a serum titer that is comparable to a wild typemouse immunized with the same antigen. In a specific embodiment, themouse expresses a single κ light chain from no more than one rearrangedκ light chain sequence. In one embodiment, the serum titer ischaracterized as total immunoglobulin. In a specific embodiment, theserum titer is characterized as IgM specific titer. In a specificembodiment, the serum titer is characterized as IgG specific titer. In amore specific embodiment, the rearranged κ light chain sequence isselected from a Vκ1-39Jκ5 and Vκ3-20Jκ1 sequence. In one embodiment, therearranged κ light chain sequence is a Vκ1-39Jκ5 sequence. In oneembodiment, the rearranged κ light chain sequence is a Vκ3-20Jκ1sequence.

In one aspect, a genetically modified mouse is provided that expresses apopulation of antigen-specific antibodies, wherein all of theimmunoglobulin light chains of the population of antigen-specificantibodies comprise a human light chain variable (V_(L)) region derivedfrom the same single human V_(L) gene segment and the immunoglobulinheavy chains comprise a human heavy chain variable (V_(H)) regionderived from one of a plurality of human V_(H) gene segments.

In various embodiments, the human V_(H) gene segments are selected fromV_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24, V_(H)1-46,V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7,V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-20, V_(H)3-21,V_(H)3-23, V_(H)3-30, V_(H)3-33, V_(H)3-43, V_(H)3-48, V_(H)3-53,V_(H)3-64, V_(H)3-72, V_(H)3-73, V_(H)4-31, V_(H)4-34, V_(H)4-39,V_(H)4-59, V_(H)5-51, and V_(H)6-1.

In various embodiments, same single human V_(L) gene segment is selectedfrom a human Vκ1-39 gene segment and a human Vκ3-20 gene segment. Invarious embodiments, all of the immunoglobulin light chains comprise ahuman light chain J (J_(L)) gene segment selected from a Jκ and a Jλgene segment. In a specific embodiment, the human J_(L) gene segment isselected from a human Jκ1 and a Jκ5 gene segment. In variousembodiments, the mouse lacks a sequence selected from a mouseimmunoglobulin V_(L) gene segment, a mouse immunoglobulin J_(L) genesegment, and a combination thereof. In various embodiments, the humanV_(L) region is operably linked to a human, mouse, or rat immunoglobulinlight chain constant (C_(L)) region. In a specific embodiment, the humanV_(L) region is operably linked to a mouse Cκ region. In a specificembodiment, the human V_(L) region is operably linked to a rat Cκregion.

In various embodiments, the human V_(L) region is expressed from anendogenous immunoglobulin light chain locus. In various embodiments, thehuman V_(H) region is operably linked to a human, mouse, or ratimmunoglobulin heavy chain constant (C_(H)) region. In variousembodiments the (C_(H)) region comprises a human sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, a C_(H)4, and/or a combinationthereof. In various embodiments, the human V_(H) region is expressedfrom an endogenous immunoglobulin heavy chain locus.

In one aspect, a genetically modified mouse is provided that expresses aplurality of immunoglobulin heavy chains associated with a single lightchain. In one embodiment, the heavy chain comprises a human sequence. Invarious embodiments, the human sequence is selected from a variablesequence, a C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combinationthereof. In one embodiment, the single light chain comprises a humansequence. In various embodiments, the human sequence is selected from avariable sequence, a constant sequence, and a combination thereof. Inone embodiment, the mouse comprises a disabled endogenous immunoglobulinlocus and expresses the heavy chain and/or the light chain from atransgene or extrachromosomal episome. In one embodiment, the mousecomprises a replacement at an endogenous mouse locus of some or allendogenous mouse heavy chain gene segments (i.e., V, D, J), and/or someor all endogenous mouse heavy chain constant sequences (e.g., C_(H)1,hinge, C_(H)2, C_(H)3, or a combination thereof), and/or some or allendogenous mouse light chain sequences (e.g., V, J, constant, or acombination thereof), with one or more human immunoglobulin sequences.

In one aspect, a mouse suitable for making antibodies that have the samelight chain is provided, wherein all or substantially all antibodiesmade in the mouse are expressed with the same light chain. In oneembodiment, the light chain is expressed from an endogenous light chainlocus.

In one aspect, a method for making a light chain for a human antibody isprovided, comprising obtaining from a mouse as described herein a lightchain sequence and a heavy chain sequence, and employing the light chainsequence and the heavy chain sequence in making a human antibody. In oneembodiment, the human antibody is a bispecific antibody.

In one aspect, a method for identifying a human heavy chain variabledomain that is capable of binding an antigen of interest with anengineered light chain as described herein is provided, wherein themethod comprises providing a heavy chain variable domain derived from afirst antibody that is capable of binding the antigen, repairing theheavy chain variable domain with a germline light chain sequence andtransfecting a cell so that each are expressed to form a secondantibody, exposing the second antibody to the antigen, and measuringbinding of the second antibody to the antigen.

In one embodiment, the light chain of the first antibody comprises ahuman Vκ1-39 sequence. In one embodiment, the light chain of the firstantibody comprises a human Vκ3-20 sequence. In one embodiment, thegermline light chain sequence comprises a human Vκ1-39 or Vκ3-20sequence. In various embodiments, binding of the second antibody to theantigen is determined by comparison of binding of the first antibody tothe antigen.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates a targeting strategy for replacing endogenous mouseimmunoglobulin light chain variable region gene segments with a humanVκ1-39Jκ5 gene region.

FIG. 2 illustrates a targeting strategy for replacing endogenous mouseimmunoglobulin light chain variable region gene segments with a humanVκ3-20Jκ1 gene region.

FIG. 3 illustrates a targeting strategy for replacing endogenous mouseimmunoglobulin light chain variable region gene segments with a humanVpreB/Jλ5 gene region.

FIG. 4 shows the percent of CD19⁺ B cells (y-axis) from peripheral bloodfor wild type mice (WT), mice homozygous for an engineered humanrearranged Vκ1-39Jκ5 light chain region (Vκ1-39Jκ5 HO) and micehomozygous for an engineered human rearranged Vκ3-20Jκ1 light chainregion (Vκ3-20Jκ1 HO).

FIG. 5A shows the relative mRNA expression (y-axis) of a Vκ1-39-derivedlight chain in a quantitative PCR assay using probes specific for thejunction of an engineered human rearranged Vκ1-39Jκ5 light chain region(Vκ1-39Jκ5 Junction Probe) and the human Vκ1-39 gene segment (Vκ1-39Probe) in a mouse homozygous for a replacement of the endogenous Nix andJκ gene segments with human Vκ and Jκ gene segments (Hκ), a wild typemouse (WT), and a mouse heterozygous for an engineered human rearrangedVκ1-39Jκ5 light chain region (Vκ1-39Jκ5 HET). Signals are normalized toexpression of mouse Cκ. N.D.: not detected.

FIG. 5B shows the relative mRNA expression (y-axis) of a Vκ1-39-derivedlight chain in a quantitative PCR assay using probes specific for thejunction of an engineered human rearranged Vκ1-39Jκ5 light chain region(Vκ1-39Jκ5 Junction Probe) and the human Vκ1-39 gene segment (Vκ1-39Probe) in a mouse homozygous for a replacement of the endogenous Nix andJκ gene segments with human Vκ and Jκ gene segments (Hκ), a wild typemouse (WT), and a mouse homozygous for an engineered human rearrangedVκ1-39Jκ5 light chain region (Vκ1-39Jκ5 HO). Signals are normalized toexpression of mouse Cκ.

FIG. 5C shows the relative mRNA expression (y-axis) of a Vκ3-20-derivedlight chain in a quantitative PCR assay using probes specific for thejunction of an engineered human rearranged Vκ3-20Jκ1 light chain region(Vκ3-20Jκ1 Junction Probe) and the human Vκ3-20 gene segment (Vκ3-20Probe) in a mouse homozygous for a replacement of the endogenous Nix andJκ gene segments with human Vκ and Jκ gene segments (Hκ), a wild typemouse (WT), and a mouse heterozygous (HET) and homozygous (HO) for anengineered human rearranged Vκ3-20Jκ1 light chain region. Signals arenormalized to expression of mouse Cκ.

FIG. 6A shows IgM (left) and IgG (right) titer in wild type (WT; N=2)and mice homozygous for an engineered human rearranged Vκ1-39Jκ5 lightchain region (Vκ1-39Jκ5 HO; N=2) immunized with β-galatosidase.

FIG. 6B shows total immunoglobulin (IgM, IgG, IgA) titer in wild type(WT; N=5) and mice homozygous for an engineered human rearrangedVκ3-20Jκ1 light chain region (Vκ3-20Jκ1 HO; N=5) immunized withβ-galatosidase.

FIG. 7A shows a schematic of monospecific antibodies (Parent-1 andParent-2) and a bispecific antibody (Bispecific) constructed from heavychain variable regions from each parent monospecific antibody. A commonlight chain variable region (darkened) is indicated in the bispecificantibody.

FIG. 7B shows a schematic for the binding characteristics of two parentmonoclonal antibodies (Parent-1 and Parent-2) for an antigen ofinterest, as well as the binding characteristic of a bispecific antibodyconstructed from pairing the heavy chain variable regions from eachmonospecific parent antibody with a common light chain. The capabilityof the bispecific antibody to bind to two distinct epitopes of theantigen of interest either separately (bottom left) or simultaneously(bottom right) is indicated.

FIG. 8 shows a bar graph of the binding of 300 nM bispecific (darkenedbars) and monospecific (striped and gray bars) antibodies to a capturedmonomeric Antigen E surface in BIACORE™ units (RU). Monoclonal parent-1antibody (P1 Ab), monoclonal parent-2 (P2 Ab) and bispecific antibodies(BsAb) are indicated.

FIG. 9 shows two genetically modified endogenous immunoglobulin lightchain (e.g., κ light chain) loci. The locus on the top (DLC-5J) containsan engineered human DNA fragment (striped line) containing two human Vκgene segments and five human Jκ gene segments. The locus on the bottom(DLC-1J) contains an engineered human DNA fragment (striped line)containing two human Vκ gene segments and one human Jκ gene segment.Each locus is capable of rearranging to form a human Vκ region operablylinked to an endogenous light chain constant region (e.g., a Cκ).Immunoglobulin promoters (arrow above locus), leader exons (closedarrows), and the two human Vκ gene segments (open arrows), all flankedupstream (5′) by a neomycin cassette containing Frt recombination sitesare shown. Recombination signal sequences engineered with each of thehuman gene segments (Vκ and Jκ) are indicated by open ovals juxtaposedwith each gene segment.

FIG. 10A, in the top panel, shows representative contour plots of bonemarrow stained for B and T cells (CD19⁺ and CD3⁺, respectively) from awild type mouse (WT) and a mouse homozygous for two human Vκ and fivehuman Jκ gene segments (DLC-5J). The bottom panel shows representativecontour plots of bone marrow gated on CD19⁺ and stained for ckit⁺ andCD43⁺ from a wild type mouse (WT) and a mouse homozygous for two humanVκ and five human Jκ gene segments (DLC-5J). Pro and Pre B cells arenoted on the contour plots of the bottom panel.

FIG. 10B shows the number of Pro (CD19⁺CD43⁺ckit⁺) and Pre(CD19⁺CD43⁻ckit⁻) B cells in bone marrow harvested from the femurs ofwild type mice (WT) and mice homozygous for two human Vκ and five humanJκ gene segments (DLC-5J).

FIG. 11A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for two human Vκ and five human Jκgene segments (DLC-5J). Immature, mature and pro/pre B cells are notedon each of the contour plots.

FIG. 11B shows the total number of B (CD19⁺), immature B(B220^(int)IgM⁺) and mature B (B220^(hi)IgM⁺) cells in bone marrowisolated from the femurs of wild type mice (WT) and mice homozygous fortwo human Vκ and five human Jκ gene segments (DLC-5J).

FIG. 12A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for two human Vκ and five human Jκgene segments (DLC-5J). Immature, mature and pro/pre B cells are notedon each of the contour plots.

FIG. 12B shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells stained forIgλ and Igκ expression isolated from the femurs of a wild type mouse(WT) and a mouse homozygous for two human Vκ and five human Jκ genesegments (DLC-5J).

FIG. 13A, in the top panel, shows representative contour plots ofsplenocytes gated on singlets and stained for B and T cells (CD19⁺ andCD3⁺, respectively) from a wild type mouse (WT) and a mouse homozygousfor two human Vκ and five human Jκ gene segments (DLC-5J). The bottompanel shows representative contour plots of splenocytes gated on CD19⁺and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from awild type mouse (WT) and a mouse homozygous for two human Vκ and fivehuman Jκ gene segments (DLC-5J). Mature (54 for WT, 56.9 for DLC-5J) andtransitional (23.6 for WT, 25.6 for DLC-5J) B cells are noted on each ofthe contour plots.

FIG. 13B shows the total number of CD19⁺ B cells, transitional B cells(CD19⁺IgM^(hi)IgD^(lo)) and mature B cells (CD19⁺IgM^(lo)IgD^(hi)) inharvested spleens from wild type mice (WT) and mice homozygous for twohuman Vκ and five human Jκ gene segments (DLC-5J).

FIG. 14A shows representative contour plots of Igλ⁺ and Igκ⁺ splenocytesgated on CD19⁺ from a wild type mouse (WT) and a mouse homozygous fortwo human Vκ and five human Jκ gene segments (DLC-5J).

FIG. 14B shows the total number of B cells (CD19⁺), Igκ⁺ B cells(CD19⁺Igκ⁺) and Igλ⁺ B cells (CD19⁺Igλ⁺) in harvested spleens from wildtype (WT) and mice homozygous for two human Vκ and five human Jκ genesegments (DLC-5J).

FIG. 15A shows the peripheral B cell development in mice homozygous fortwo human Vκ and five human Jκ gene segments. The first (far left)contour plot shows CD93⁺ and B220⁺ splenocytes gated on CD19⁺ indicatingimmature (39.6) and mature (57.8) B cells. The second (top middle)contour plot shows IgM⁺ and CD23⁺ expression in immature B cellsindicating T1 (33.7; IgD⁻IgM⁺CD21^(lo)CD23⁻), T2 (21.2;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) and T3 (29.1) B cell populations. Thethird (bottom middle) contour plot shows CD21⁺ (CD35⁺) and IgM⁺expression of mature B cells indicating a small population (14.8) whichgive rise to marginal zone B cells and a second population (70.5) whichgives rise to follicular (FO) B cells. The fourth (top right) contourplot shows B220⁺ and CD23⁺ expression in mature B cells indicatingmarginal zone (90.5; MZ) and marginal zone precursor (7.3;IgM^(hi)IgD^(hi)CD21^(hi)CD23⁺) B cell populations. The fifth (bottomright) contour plot shows IgD⁺ and IgM⁺ expression in mature B cellsindicating FO-I (79.0; IgD^(hi)IgM^(hi)D21^(mid)CD23⁺) and FO-II (15.1;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) B cell populations. Percentage of cellswithin each gated region is shown.

FIG. 15B shows the peripheral B cell development in wild type mice. Thefirst (far left) contour plot shows CD93⁺ and B220⁺ splenocytes gated onCD19⁺ indicating immature (31.1) and mature (64.4) B cells. The second(top middle) contour plot shows IgM⁺ and CD23⁺ expression in immature Bcells indicating T1 (28.5; IgD⁻IgM⁺CD21^(lo)CD23⁻), T2 (28.7;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) and T3 (30.7) B cell populations. Thethird (bottom middle) contour plot shows CD21⁺ (CD35⁺) and IgM⁺expression of mature B cells indicating a small population (7.69) whichgive rise to marginal zone B cells and a second population (78.5) whichgives rise to follicular (FO) B cells. The fourth (top right) contourplot shows B220⁺ and CD23⁺ expression in mature B cells indicatingmarginal zone (79.9; MZ) and marginal zone precursor (19.4;IgM^(hi)IgD^(hi)CD21^(hi)CD23⁺) B cell populations. The fifth (bottomright) contour plot shows IgD⁺ and IgM⁺ expression in mature B cellsindicating FO-I (83.6; IgD^(hi)IgM^(lo)CD21^(mid)CD23⁺) and FO-II (13.1;IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) B cell populations. Percentage of cellswithin each gated region is shown.

FIG. 16 shows the total number of transitional, marginal zone andfollicular B cell populations in harvested spleens of wild-type (WT) andmice homozygous for two human Vκ and five human Jκ gene segments(DLC-5J).

FIG. 17 shows the relative mRNA expression in bone marrow (y-axis) ofVκ3-20-derived and Vκ1-39-derived light chains in a quantitative PCRassay using probes specific for Vκ3-20 or Vκ1-39 gene segments in micehomozygous for a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (Hκ), wild type mice (WT), micehomozygous for two human Vκ gene segments and five human Jκ genesegments (DLC-5J) and mice homozygous for two human Vκ gene segments andone human Jκ gene segment (DLC-1J). Signals are normalized to expressionof mouse Cκ. ND: not detected.

FIG. 18 shows the relative mRNA expression in whole spleens (y-axis) ofVκ3-20-derived and Vκ1-39-derived light chains in a quantitative PCRassay using probes specific for Vκ3-20 or Vκ1-39 gene segments in micehomozygous for a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (Hκ), wild type mice (WT), micehomozygous for two human Vκ gene segments and five human Jκ genesegments (DLC-5J) and mice homozygous for two human Vκ gene segments andone human Jκ gene segment (DLC-1J). Signals are normalized to expressionof mouse Cκ. ND: not detected.

FIG. 19 shows a general illustration of recombination of a V and a Jgene segment of an immunoglobulin κ light chain allele in a mouse andthe structure of the light chain locus before rearrangement (top) andafter rearrangement (bottom). Such a rearrangement as shown is only oneof several possible rearrangement events.

DETAILED DESCRIPTION

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference in their entirety.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable (V_(H)) region and a heavy chainconstant region (C_(H)). The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable (V_(L)) region and a light chain constant region (C_(L)).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) comprises three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may beabbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may beabbreviated as LCDR1, LCDR2 and LCDR3. The term “high affinity” antibodyrefers to an antibody that has a K_(D) with respect to its targetepitope about of 10⁻⁹ M or lower (e.g., about 1×10⁻⁹ M, 1×10⁻¹⁰ M,1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment, K_(D) is measured bysurface plasmon resonance, e.g., BIACORE™; in another embodiment, K_(D)is measured by ELISA.

The phrase “bispecific antibody” refers to an antibody capable ofselectively binding two or more epitopes. Bispecific antibodies includefragments of two different monoclonal antibodies (FIG. 7A) and generallycomprise two nonidentical heavy chains derived from the two differentmonoclonal antibodies, with each heavy chain specifically binding adifferent epitope—either on two different molecules (e.g., differentepitopes on two different immunogens; see FIG. 7B, bottom left) or onthe same molecule (e.g., different epitopes on the same immunogen; seeFIG. 7B, bottom right). If a bispecific antibody is capable ofselectively binding two different epitopes (a first epitope and a secondepitope), the affinity of the first heavy chain for the first epitopewill generally be at least one to two or three or four or more orders ofmagnitude lower than the affinity of the first heavy chain for thesecond epitope, and vice versa. Epitopes specifically bound by thebispecific antibody can be on the same or a different target (e.g., onthe same or a different protein; see FIG. 7B). Exemplary bispecificantibodies include those with a first heavy chain specific for a tumorantigen and a second heavy chain specific for a cytotoxic marker, e.g.,an Fc receptor (e.g., FcγRI, FcγRII, FcγRIII, etc.) or a T cell marker(e.g., CD3, CD28, etc.). Further, the second heavy chain variable regioncan be substituted with a heavy chain variable region having a differentdesired specificity. For example, a bispecific antibody with a firstheavy chain specific for a tumor antigen and a second heavy chainspecific for a toxin can be paired so as to deliver a toxin (e.g.,saporin, vinca alkaloid, etc.) to a tumor cell. Other exemplarybispecific antibodies include those with a first heavy chain specificfor an activating receptor (e.g., B cell receptor, FcγRI, FcγRIIA,FcγRIIIA, FcαRI, T cell receptor, etc.) and a second heavy chainspecific for an inhibitory receptor (e.g., FcγRIIB, CD5, CD22, CD72,CD300a, etc.). Such bispecific antibodies can be constructed fortherapeutic conditions associated with cell activation (e.g. allergy andasthma). Bispecific antibodies can be made, for example, by combiningheavy chains that recognize different epitopes of the same or differentimmunogen (FIG. 7B). For example, nucleic acid sequences encoding heavychain variable sequences that recognize different epitopes of the sameor different immunogen can be fused to nucleic acid sequences encodingthe same or different heavy chain constant regions, and such sequencescan be expressed in a cell that expresses an immunoglobulin light chain.A typical bispecific antibody has two heavy chains each having threeheavy chain CDRs, followed by (N-terminal to C-terminal) a C_(H)1domain, a hinge, a C_(H)2 domain, and a C_(H)3 domain, and animmunoglobulin light chain that either does not confer epitope-bindingspecificity but that can associate with each heavy chain, or that canassociate with each heavy chain and that can bind one or more of theepitopes bound by the heavy chain epitope-binding regions, or that canassociate with each heavy chain and enable binding or one or both of theheavy chains to one or both epitopes.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g., aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. A CDR can be somatically mutated(e.g., vary from a sequence encoded in an animal's germline), humanized,and/or modified with amino acid substitutions, additions, or deletions.In some circumstances (e.g., for a CDR3), CDRs can be encoded by two ormore sequences (e.g., germline sequences) that are not contiguous (e.g.,in an unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as the result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of avariable region to specifically bind a target epitope with a desiredaffinity. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be substitution of any native residue in a protein withalanine, as used in, for example, alanine scanning mutagenesis. In someembodiments, a conservative substitution is made that has a positivevalue in the PAM250 log-likelihood matrix disclosed in Gonnet et al.(1992) Exhaustive Matching of the Entire Protein Sequence Database,Science 256:1443-45, hereby incorporated by reference. In someembodiments, the substitution is a moderately conservative substitutionwherein the substitution has a nonnegative value in the PAM250log-likelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chainor heavy chain differ by one or more conservative amino acidsubstitutions. In some embodiments, residue positions in animmunoglobulin light chain or functional fragment thereof (e.g., afragment that allows expression and secretion from, e.g., a B cell) arenot identical to a light chain whose amino acid sequence is listedherein, but differs by one or more conservative amino acidsubstitutions.

The phrase “epitope-binding protein” includes a protein having at leastone CDR and that is capable of selectively recognizing an epitope, e.g.,is capable of binding an epitope with a K_(D) that is at about onemicromolar or lower (e.g., a K_(D) that is about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁹ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).Therapeutic epitope-binding proteins (e.g., therapeutic antibodies)frequently require a K_(D) that is in the nanomolar or the picomolarrange.

The phrase “functional fragment” includes fragments of epitope-bindingproteins that can be expressed, secreted, and specifically bind to anepitope with a K_(D) in the micromolar, nanomolar, or picomolar range.Specific recognition includes having a K_(D) that is at least in themicromolar range, the nanomolar range, or the picomolar range.

The term “germline” includes reference to an immunoglobulin nucleic acidsequence in a non-somatically mutated cell, e.g., a non-somaticallymutated B cell or pre-B cell or hematopoietic cell.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain constant region sequence from any organism.Heavy chain variable domains include three heavy chain CDRs and four FRregions, unless otherwise specified. Fragments of heavy chains includeCDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has,following the variable domain (from N-terminal to C-terminal), a C_(H)1domain, a hinge, a C_(H)2 domain, and a C_(H)3 domain. A functionalfragment of a heavy chain includes a fragment that is capable ofspecifically recognizing an epitope (e.g., recognizing the epitope witha K_(D) in the micromolar, nanomolar, or picomolar range), that iscapable of expressing and secreting from a cell, and that comprises atleast one CDR.

The term “identity” when used in connection with sequence includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences, but in the case of a light chainconstant domain, the length should contain sequence of sufficient lengthto fold into a light chain constant domain that is capable ofself-association to form a canonical light chain constant domain, e.g.,capable of forming two beta sheets comprising beta strands and capableof interacting with at least one C_(H)1 domain of a human or a mouse. Inthe case of a C_(H)1 domain, the length of sequence should containsequence of sufficient length to fold into a C_(H)1 domain that iscapable of forming two beta sheets comprising beta strands and capableof interacting with at least one light chain constant domain of a mouseor a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human κ and λlight chains and a VpreB, as well as surrogate light chains. Light chainvariable (V_(L)) domains typically include three light chain CDRs andfour framework (FR) regions, unless otherwise specified. Generally, afull-length light chain includes, from amino terminus to carboxylterminus, a V_(L) domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,and a light chain constant domain. Light chains include those, e.g.,that do not selectively bind either a first or a second epitopeselectively bound by the epitope-binding protein in which they appear.Light chains also include those that bind and recognize, or assist theheavy chain with binding and recognizing, one or more epitopesselectively bound by the epitope-binding protein in which they appear.Common light chains are those derived from a rearranged human Vκ1-39Jκ5sequence or a rearranged human Vκ3-20Jκ1 sequence, and includesomatically mutated (e.g., affinity matured) versions.

The phrase “micromolar range” is intended to mean 1-999 micromolar; thephrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase“picomolar range” is intended to mean 1-999 picomolar.

The phrase “somatically mutated” includes reference to a nucleic acidsequence from a B cell that has undergone class-switching, wherein thenucleic acid sequence of an immunoglobulin variable region (e.g., aheavy chain variable domain or including a heavy chain CDR or FRsequence) in the class-switched B cell is not identical to the nucleicacid sequence in the B cell prior to class-switching, such as, forexample, a difference in a CDR or framework nucleic acid sequencebetween a B cell that has not undergone class-switching and a B cellthat has undergone class-switching. “Somatically mutated” includesreference to nucleic acid sequences from affinity-matured B cells thatare not identical to corresponding immunoglobulin variable regionsequences in B cells that are not affinity-matured (i.e., sequences inthe genome of germline cells). The phrase “somatically mutated” alsoincludes reference to an immunoglobulin variable region nucleic acidsequence from a B cell after exposure of the B cell to an epitope ofinterest, wherein the nucleic acid sequence differs from thecorresponding nucleic acid sequence prior to exposure of the B cell tothe epitope of interest. The phrase “somatically mutated” refers tosequences from antibodies that have been generated in an animal, e.g., amouse having human immunoglobulin variable region nucleic acidsequences, in response to an immunogen challenge, and that result fromthe selection processes inherently operative in such an animal.

The term “unrearranged,” with reference to a nucleic acid sequence,includes nucleic acid sequences that exist in the germline of an animalcell.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

Universal Light Chain

Prior efforts to make useful multispecific epitope-binding proteins,e.g., bispecific antibodies, have been hindered by variety of problemsthat frequently share a common paradigm: in vitro selection ormanipulation of sequences to rationally engineer, or to engineer throughtrial-and-error, a suitable format for pairing a heterodimericbispecific human immunoglobulin. Unfortunately, most if not all of thein vitro engineering approaches provide largely ad hoc fixes that aresuitable, if at all, for individual molecules. On the other hand, invivo methods for employing complex organisms to select appropriatepairings that are capable of leading to human therapeutics have not beenrealized.

Mice containing human immunoglobulin loci, variable and constant regionsrandomly inserted into the mouse genome, are known in the art. Initialstrains of such mice contained a limited number of human immunoglobulingene segments. Specifically, a handful of strains containing humanimmunoglobulin light chain gene segments contained either one, three orfour human immunoglobulin V_(L) gene segments and five humanimmunoglobulin J_(L) gene segments (Taylor et al. 1992, Nucleic AcidsResearch 20(23): 6287-6295; Fishwild et al. 1996, Nature Biotechnology14: 845-851; Lonberg et al. 1994, Nature 368: 856-859; Green et al.1994, Nature Genetics 7:13-21; Green and Jakobovits 1998, J. Exp. Med.188(3): 483-495; Green 1999, J. Immunol. Methods 231: 11-23). These micethat contained only a few human immunoglobulin V_(L) gene segments aspart of fully human transgenes randomly inserted into the mouse genomedemonstrated compromised B cell numbers, impaired B cell development andother immune deficiencies. Expression of the human immunoglobulin V_(L)genes, as detected by surface expression of human Cκ on B cells, waslower than the endogenous κ light chain as compared to wild type.Surprisingly, the present invention provides mice whose B cell numbersand development is nearly wild-type in respects when mice are engineeredat the endogenous immunoglobulin κ light chain loci to contain eitherone or two human immunoglobulin Vκ gene segments (e.g., Examples 2 and14, Tables 3, 25 and 26, and FIGS. 4, 10A-18). Further, in someembodiments, mice provided by the present invention, are able togenerate several high-affinity reverse chimeric antibodies containinghuman V_(H) and V_(L) domains in response to antigen, wherein the V_(L)domains each contain one of two possible human V_(L) gene segments andone of five possible human J_(L) gene segments (e.g., see Examples 5-10,12, and 14). Thus, in contrast to preliminary strains of mice engineeredwith human immunoglobulin light chain miniloci (i.e., a limited numberof human immunoglobulin gene segments), presently provided engineeredmice that contain a limited number of human immunoglobulin V_(L) genesegments (either one or two) and, in some embodiments, two or more(e.g., 2, 3, 4, or 5) human immunoglobulin J_(L) gene segments,surprisingly exhibit normal B cell numbers, normal immunoglobulin lightchain expression, and normal B cell development. Further, such providedmice also show no reduced or impaired ability to mount robust immuneresponses to multiple antigens as a result of a limited immunoglobulinlight chain repertoire. Accordingly, mice are provided that comprise ahumanized V_(L) locus comprising no more than two unrearranged humanimmunoglobulin V_(L) gene segments and two or more (e.g., 2, 3, 4, or 5)human immunoglobulin J_(L) gene segments—or no more than two rearrangedhuman V_(L)J_(L) segments—and that exhibit wild-type B cell populationsin number, and exhibit wild-type B cell development.

Generally, native mouse sequences are frequently not a good source forhuman therapeutic sequences. For at least that reason, generating mouseheavy chain immunoglobulin variable regions that pair with a commonhuman light chain is of limited practical utility. More in vitroengineering efforts would be expended in a trial-and-error process totry to humanize the mouse heavy chain variable sequences while hoping toretain epitope specificity and affinity while maintaining the ability tocouple with the common human light chain, with uncertain outcome. At theend of such a process, the final product may maintain some of thespecificity and affinity, and associate with the common light chain, butultimately immunogenicity in a human would likely remain a profoundrisk.

Therefore, a suitable mouse for making human therapeutics would includea suitably large repertoire of human heavy chain variable region genesegments in place of endogenous mouse heavy chain variable region genesegments. The human heavy chain variable region gene segments should beable to rearrange and recombine with an endogenous mouse heavy chainconstant domain to form a reverse chimeric heavy chain (i.e., a heavychain comprising a human variable domain and a mouse constant region).The heavy chain should be capable of class switching and somatichypermutation so that a suitably large repertoire of heavy chainvariable domains are available for the mouse to select one that canassociate with the limited repertoire of human light chain variableregions.

A mouse that selects a common light chain for a plurality of heavychains has a practical utility. In various embodiments, antibodies thatexpress in a mouse that can only express a common light chain will haveheavy chains that can associate and express with an identical orsubstantially identical light chain. This is particularly useful inmaking bispecific antibodies. For example, such a mouse can be immunizedwith a first immunogen to generate a B cell that expresses an antibodythat specifically binds a first epitope. The mouse (or a mousegenetically the same) can be immunized with a second immunogen togenerate a B cell that expresses an antibody that specifically binds thesecond epitope. Variable heavy chain regions can be cloned from the Bcells and expressed with the same heavy chain constant region, and thesame variable light chain region (e.g., a common light chain) in a cellto make a bispecific antibody, wherein the variable heavy chaincomponent of the bispecific antibody has been selected by a mouse toassociate and express with the variable light chain (or common lightchain) component.

The inventors have engineered a mouse for generating immunoglobulinlight chains that will suitably pair with a rather diverse family ofheavy chains, including heavy chains whose variable regions depart fromgermline sequences, e.g., affinity matured or somatically mutatedvariable regions. In various embodiments, the mouse is devised to pairhuman light chain variable domains with human heavy chain variabledomains that comprise somatic mutations, thus enabling a route to highaffinity binding proteins suitable for use as human therapeutics.

The genetically engineered mouse, through the long and complex processof antibody selection within an organism, makes biologically appropriatechoices in pairing a diverse collection of human heavy chain variabledomains with a limited number of human light chain options. In order toachieve this, the mouse is engineered to present a limited number ofhuman light chain variable domain options in conjunction with a widediversity of human heavy chain variable domain options. Upon challengewith an immunogen, the mouse maximizes the number of solutions in itsrepertoire to develop an antibody to the immunogen, limited largely orsolely by the number or light chain options in its repertoire. Invarious embodiments, this includes allowing the mouse to achievesuitable and compatible somatic mutations of the light chain variabledomain that will nonetheless be compatible with a relatively largevariety of human heavy chain variable domains, including in particularsomatically mutated human heavy chain variable domains.

To achieve a limited repertoire of light chain options, the mouse isengineered to render nonfunctional or substantially nonfunctional itsability to make, or rearrange, a native mouse light chain variabledomain. This can be achieved, e.g., by deleting the mouse's light chainvariable region gene segments. The endogenous mouse locus can then bemodified by an exogenous suitable human light chain variable region genesegment of choice, operably linked to the endogenous mouse light chainconstant domain, in a manner such that the exogenous human variableregion gene segments can combine with the endogenous mouse light chainconstant region gene and form a rearranged reverse chimeric light chaingene (human variable, mouse constant). In various embodiments, the lightchain variable region is capable of being somatically mutated. Invarious embodiments, to maximize ability of the light chain variableregion to acquire somatic mutations, the appropriate enhancer(s) isretained in the mouse. For example, in modifying a mouse κ light chainlocus to replace endogenous mouse κ light chain gene segments with humanκ light chain gene segments, the mouse κ intronic enhancer and mouse κ3′ enhancer are functionally maintained, or undisrupted.

A genetically engineered mouse is provided that expresses a limitedrepertoire of reverse chimeric (human variable, mouse constant) lightchains associated with a diversity of reverse chimeric (human variable,mouse constant) heavy chains. In various embodiments, the endogenousmouse κ light chain gene segments are deleted and replaced with a single(or two) rearranged human light chain region, operably linked to theendogenous mouse Cκ gene. In embodiments for maximizing somatichypermutation of the rearranged human light chain region, the mouse κintronic enhancer and the mouse κ 3′ enhancer are maintained. In variousembodiments, the mouse also comprises a nonfunctional λ light chainlocus, or a deletion thereof or a deletion that renders the locus unableto make a λ light chain.

A genetically engineered mouse is provided that, in various embodiments,comprises a light chain variable region locus lacking endogenous mouselight chain V_(L) and J_(L) gene segments and comprising a rearrangedhuman light chain variable region, in one embodiment a rearranged humanV_(L)/J_(L) sequence, operably linked to a mouse constant region,wherein the locus is capable of undergoing somatic hypermutation, andwherein the locus expresses a light chain comprising the humanV_(L)/J_(L) sequence linked to a mouse constant region. Thus, in variousembodiments, the locus comprises a mouse κ 3′ enhancer, which iscorrelated with a normal, or wild type, level of somatic hypermutation.

The genetically engineered mouse in various embodiments when immunizedwith an antigen of interest generates B cells that exhibit a diversityof rearrangements of human immunoglobulin heavy chain variable regionsthat express and function with one or with two rearranged light chains,including embodiments where the one or two light chains comprise humanlight chain variable regions that comprise, e.g., 1 to 5 somaticmutations. In various embodiments, the human light chains so expressedare capable of associating and expressing with any human immunoglobulinheavy chain variable region expressed in the mouse.

In addition to genetically engineered mice comprising restrictedimmunoglobulin light chain repertoire (e.g., a single human V_(L) genesegment or no more than two human V_(L) gene segments and, one humanJ_(L) gene segment or, optionally, two or more human J_(L) genesegments) as described herein, also provided herein are othergenetically modified non-human animals that comprise a single humanV_(L) gene segment or no more than two human V_(L) gene segments. Insome embodiments, such non-human animals comprise a single rearrangedhuman V_(L) region composed of a rearranged human V_(L)J_(L) sequence.In some embodiments, such non-human animals comprise no more than twohuman V_(L) gene segments and two or more (e.g., 2, 3, 4, or 5 humanJ_(L) gene segments. In various embodiments, human gene segments areoperably linked to a non-human light chain constant region, e.g., amouse a rat light chain constant region.

Such non-human animals may be selected from a group consisting of amouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep,goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesusmonkey). For the non-human animals where suitable genetically modifiableES cells are not readily available, other methods are employed to make anon-human animal comprising genetic modifications as described herein.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

In some embodiments, a non-human animal of the present invention is amammal. In some embodiments, a non-human animal of the present inventionis a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. Insome embodiments, a genetically modified animal of the present inventionis a rodent. In some embodiments, a rodent of the present invention isselected from a mouse, a rat, and a hamster. In some embodiments, arodent of the present invention is selected from the superfamilyMuroidea. In some embodiments, a genetically modified animal of thepresent invention is from a family selected from Calomyscidae (e.g.,mouse-like hamsters), Cricetidae (e.g., hamster, New World rats andmice, voles), Muridae (true mice and rats, gerbils, spiny mice, crestedrats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasyrats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rates, bamboo rats, and zokors). In some certainembodiments, a genetically modified rodent of the present invention isselected from a true mouse or rat (family Muridae), a gerbil, a spinymouse, and a crested rat. In some certain embodiments, a geneticallymodified mouse of the present invention is from a member of the familyMuridae. In some embodiment, an non-human animal of the presentinvention is a rodent. In some certain embodiments, a rodent of thepresent invention is selected from a mouse and a rat. In someembodiments, a non-human animal of the present invention is a mouse.

In some embodiments, a non-human animal of the present invention is arodent that is a mouse of a C57BL strain selected from C57BL/A,C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In somecertain embodiments, a mouse of the present invention is a 129 strainselected from the group consisting of a strain that is 129P1, 129P2,129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5,129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g.,Festing et al., 1999, Mammalian Genome 10:836; Auerbach et al., 2000,Biotechniques 29(5):1024-1028, 1030, 1032). In some certain embodiments,a genetically modified mouse of the present invention is a mix of anaforementioned 129 strain and an aforementioned C57BL/6 strain. In somecertain embodiments, a mouse of the present invention is a mix ofaforementioned 129 strains, or a mix of aforementioned BL/6 strains. Insome certain embodiments, a 129 strain of the mix as described herein isa 129S6 (129/SvEvTac) strain. In some embodiment, a mouse of the presentinvention is a BALB strain, e.g., BALB/c strain. In some embodiments, amouse of the present invention is a mix of a BALB strain and anotheraforementioned strain.

In some embodiments, a non-human animal of the present invention is arat. In some certain embodiments, a rat of the present invention isselected from a Wistar rat, an LEA strain, a Sprague Dawley strain, aFischer strain, F344, F6, and Dark Agouti. In some certain embodiments,a rat strain as described herein is a mix of two or more strainsselected from the group consisting of Wistar, LEA, Sprague Dawley,Fischer, F344, F6, and Dark Agouti.

Epitope-Binding Proteins Binding More than One Epitope

Compositions and methods described herein can be used to make bindingproteins that bind more than one epitope with high affinity, e.g.,bispecific antibodies. Advantages of the invention include the abilityto select suitably high binding (e.g., affinity matured) heavy chainimmunoglobulin chains each of which will associate with a single lightchain.

Several techniques for making bispecific antibody fragments fromrecombinant cell culture have been reported. However, synthesis andexpression of bispecific binding proteins has been problematic, in partdue to issues associated with identifying a suitable light chain thatcan associate and express with two different heavy chains, and in partdue to isolation issues. In various embodiments, compositions andmethods described herein provide the advantage of full length bispecificantibodies that do not require special modification(s) to maintaintraditional immunoglobulin structure by increasing stability/interactionof the components (FIG. 7A). In various embodiments, suchmodification(s) has proven cumbersome and served as an obstacle todevelopment of bispecific antibody technology and their potential use intreating for human disease. Thus, in various embodiments, throughproviding a natural immunoglobulin structure (i.e., full length) havingthe added property of multiple specificities, full length bispecificantibodies maintain their critical effector functions that previousbispecific fragments lack, and further provide therapeutics thatdemonstrate the important pharmacokinetic parameter of a longerhalf-life.

Methods and compositions described herein allow for a geneticallymodified mouse to select, through otherwise natural processes, asuitable light chain that can associate and express with more than oneheavy chain, including heavy chains that are somatically mutated (e.g.,affinity matured). Human V_(L) and V_(H) sequences from suitable B cellsof immunized mice as described herein that express affinity maturedantibodies having reverse chimeric heavy chains (i.e., human variableand mouse constant) can be identified and cloned in frame in anexpression vector with a suitable human constant region gene sequence(e.g., a human IgG1). Two such constructs can be prepared, wherein eachconstruct encodes a human heavy chain variable domain that binds adifferent epitope. One of the human Ws (e.g., human Vκ1-39Jκ5 or humanVκ3-20Jκ1), in germline sequence or from a B cell wherein the sequencehas been somatically mutated, can be fused in frame to a suitable humanconstant region gene (e.g., a human κ constant gene). These three fullyhuman heavy and light constructs can be placed in a suitable cell forexpression. The cell will express two major species: a homodimeric heavychain with the identical light chain, and a heterodimeric heavy chainwith the identical light chain. To allow for a facile separation ofthese major species, one of the heavy chains is modified to omit aProtein A-binding determinant, resulting in a differential affinity of ahomodimeric binding protein from a heterodimeric binding protein.Compositions and methods that address this issue are described in U.S.Ser. No. 12/832,838, filed 25 Jun. 2010, entitled “Readily IsolatedBispecific Antibodies with Native Immunoglobulin Format,” published asUS 2010/0331527A1, hereby incorporated by reference.

In one aspect, an epitope-binding protein as described herein isprovided, wherein human V_(L) and V_(H) sequences are derived from micedescribed herein that have been immunized with an antigen comprising anepitope of interest.

In one embodiment, an epitope-binding protein is provided that comprisesa first and a second polypeptide, the first polypeptide comprising, fromN-terminal to C-terminal, a first epitope-binding region thatselectively binds a first epitope, followed by a constant region thatcomprises a first C_(H)3 region of a human IgG selected from IgG1, IgG2,IgG4, and a combination thereof; and, a second polypeptide comprising,from N-terminal to C-terminal, a second epitope-binding region thatselectively binds a second epitope, followed by a constant region thatcomprises a second C_(H)3 region of a human IgG selected from IgG1,IgG2, IgG4, and a combination thereof, wherein the second C_(H)3 regioncomprises a modification that reduces or eliminates binding of thesecond C_(H)3 domain to protein A.

In one embodiment, the second C_(H)3 region comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In anotherembodiment, the second C_(H)3 region further comprises a Y96Fmodification (IMGT; Y436F by EU).

In one embodiment, the second C_(H)3 region is from a modified humanIgG1, and further comprises a modification selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second C_(H)3 region is from a modified humanIgG2, and further comprises a modification selected from the groupconsisting of N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I byEU).

In one embodiment, the second C_(H)3 region is from a modified humanIgG4, and further comprises a modification selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT; Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

One method for making an epitope-binding protein that binds more thanone epitope is to immunize a first mouse in accordance with theinvention with an antigen that comprises a first epitope of interest,wherein the mouse comprises an endogenous immunoglobulin light chainvariable region locus that does not contain an endogenous mouse V_(L)that is capable of rearranging and forming a light chain, wherein at theendogenous mouse immunoglobulin light chain variable region locus is asingle rearranged human V_(L) region operably linked to the mouseendogenous light chain constant region gene, and the rearranged humanV_(L) region is selected from a human Vκ1-39Jκ5 and a human Vκ3-20Jκ1,and the endogenous mouse V_(H) gene segments have been replaced in wholeor in part with human V_(H) gene segments, such that immunoglobulinheavy chains made by the mouse are solely or substantially heavy chainsthat comprise human variable domains and mouse constant domains. Whenimmunized, such a mouse will make a reverse chimeric antibody,comprising only one of two human light chain variable domains (e.g., oneof human Vκ1-39Jκ5 or human Vκ3-20Jκ1). Once a B cell is identified thatencodes a V_(H) that binds the epitope of interest, the nucleotidesequence of the V_(H) (and, optionally, the V_(L)) can be retrieved(e.g., by PCR) and cloned into an expression construct in frame with asuitable human immunoglobulin constant domain. This process can berepeated to identify a second V_(H) domain that binds a second epitope,and a second V_(H) gene sequence can be retrieved and cloned into anexpression vector in frame to a second suitable immunoglobulin constantdomain. The first and the second immunoglobulin constant domains can thesame or different isotype, and one of the immunoglobulin constantdomains (but not the other) can be modified as described herein or in US2010/0331527A1, and epitope-binding protein can be expressed in asuitable cell and isolated based on its differential affinity forProtein A as compared to a homodimeric epitope-binding protein, e.g., asdescribed in US 2010/0331527A1.

In one embodiment, a method for making a bispecific epitope-bindingprotein is provided, comprising identifying a first affinity-matured(e.g., comprising one or more somatic hypermutations) human V_(H)nucleotide sequence (V_(H)1) from a mouse as described herein,identifying a second affinity-matured (e.g., comprising one or moresomatic hypermutations) human V_(H) nucleotide sequence (V_(H)2) from amouse as described herein, cloning V_(H)1 in frame with a human heavychain lacking a Protein A-determinant modification as described in US2010/0331527A1 for form heavy chain 1 (HC1), cloning V_(H)2 in framewith a human heavy chain comprising a Protein A-determinant as describedin US 2010/0331527A1 to form heavy chain 2 (HC2), introducing anexpression vector comprising HC1 and the same or a different expressionvector comprising HC2 into a cell, wherein the cell also expresses ahuman immunoglobulin light chain that comprises a human Vκ1-39/human Jκ5or a human Vκ3-20/human Jκ1 fused to a human light chain constantdomain, allowing the cell to express a bispecific epitope-bindingprotein comprising a V_(H) domain encoded by V_(H)1 and a V_(H) domainencoded by V_(H)2, and isolating the bispecific epitope-binding proteinbased on its differential ability to bind Protein A as compared with amonospecific homodimeric epitope-binding protein. In a specificembodiment, HC1 is an IgG1, and HC2 is an IgG1 that comprises themodification H95R (IMGT; H435R by EU) and further comprises themodification Y96F (IMGT; Y436F by EU). In one embodiment, the VH domainencoded by V_(H)1, the V_(H) domain encoded by V_(H)2, or both, aresomatically mutated.

Human V_(H) Genes That Express with a Common Human V_(L)

A variety of human variable regions from affinity-matured antibodiesraised against four different antigens were expressed with either theircognate light chain, or at least one of a human light chain selectedfrom human Vκ1-39/Jκ5, human Vκ3-20/Jκ1, or human VpreB/Jλ5 (see Example1). For antibodies to each of the antigens, somatically mutated highaffinity heavy chains from different gene families paired successfullywith rearranged human germline Vκ1-39 Jκ5 and Vκ3-20Jκ1 regions and weresecreted from cells expressing the heavy and light chains. For Vκ1-39Jκ5and Vκ3-20Jκ1, V_(H) domains derived from the following human V_(H) genefamilies expressed favorably: 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13,3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, and 6-1.Thus, a mouse that is engineered to express a limited repertoire ofhuman V_(L) domains from one or both of Vκ1-39Jκ5 and Vκ3-20Jκ1 willgenerate a diverse population of somatically mutated human V_(H) domainsfrom a V_(H) locus modified to replace mouse V_(H) gene segments withhuman V_(H) gene segments.

Mice genetically engineered to express reverse chimeric (human variable,mouse constant) immunoglobulin heavy chains associated with a singlerearranged light chain (e.g., a Vκ1-39/J or a Vκ3-20/J), when immunizedwith an antigen of interest, generated B cells that comprised adiversity of human V_(H) rearrangements and expressed a diversity ofhigh-affinity antigen-specific antibodies with diverse properties withrespect to their ability to block binding of the antigen to its ligand,and with respect to their ability to bind variants of the antigen (seeExamples 5 through 10).

Thus, the mice and methods described herein are useful in making andselecting human immunoglobulin heavy chain variable domains, includingsomatically mutated human heavy chain variable domains, that result froma diversity of rearrangements, that exhibit a wide variety of affinities(including exhibiting a K_(D) of about a nanomolar or less), a widevariety of specificities (including binding to different epitopes of thesame antigen), and that associate and express with the same orsubstantially the same human immunoglobulin light chain variable region.

Fully Human Bispecific Antibodies Having a Common Light Chain

As a first step in various embodiments, the first and second nucleicacid sequences that each encode human heavy chain variable domains (andany additional nucleic acid sequences forming the bispecific antibody)are selected from parent monoclonal antibodies having desiredcharacteristics such as, for example, capable of binding differentepitopes (see FIGS. 7A and 7B), having different affinities, etc.Normally, the nucleic acid sequences encoding the human heavy chainvariable domains are isolated from immunized mice, as described herein,to allow for fusing with human heavy chain constant regions to besuitable for human administration. Further modifications to thesequence(s) can be made by introducing mutations that add additionalfunctionality to the bispecific antibody can be achieved, which include,for example, increasing serum half-life (e.g., see U.S. Pat. No.7,217,797) and/or increasing antibody-dependent cell-mediatedcytotoxicity (e.g., see U.S. Pat. No. 6,737,056). Introducing mutationsinto the constant regions of antibodies is known in the art.Additionally, part of the bispecific antibody can be made recombinantlyin cell culture and other part(s) of the molecule can be made by thosetechniques mentioned above.

Several techniques for the producing antibodies have been described. Forexample, in various embodiments chimeric antibodies are produced in miceas described herein. Antibodies can be isolated directly from B cells ofan immunized mouse (e.g., see U.S. 2007/0280945A1) and/or the B cells ofthe immunized mouse can be used to make hybridomas (Kohler and Milstein,1975, Nature 256:495-497). DNA encoding the antibodies (human heavyand/or light chains) from mice as described herein is readily isolatedand sequenced using conventional techniques. Hybridoma and/or B cells ofderived from mice as described herein serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into host cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the murine sequences.

In various embodiments, following isolation of the DNA and selection ofthe first and second nucleic acid sequences that encode the first andsecond human heavy chain variable domains having the desiredspecificities/affinities, and a third nucleic acid sequence that encodesa human light chain domain (a germline rearranged sequence or a lightchain sequence isolated from a mouse as described herein), the threenucleic acids sequences encoding the molecules are expressed to form thebispecific antibody using recombinant techniques which are widelyavailable in the art. Often, the expression system of choice willinvolve a mammalian cell expression vector and host so that thebispecific antibody is appropriately glycosylated (e.g., in the case ofbispecific antibodies comprising antibody domains which areglycosylated). However, the molecules can also be produced in theprokaryotic expression systems. Normally, the host cell will betransformed with DNA encoding both the first human heavy chain variabledomain, the second human heavy chain variable domain, the human lightchain domain on a single vector or independent vectors. However, it ispossible to express the first human heavy chain variable domain, secondhuman heavy chain variable domain, and human light chain domain (thebispecific antibody components) in independent expression systems andcouple the expressed polypeptides in vitro. In various embodiments, thehuman light chain domain comprises a germline sequence. In variousembodiments, the human light chain domain comprises no more than one, nomore than two, no more than three, no more than four, or no more thanfive somatic hypermutations with the light chain variable sequence ofthe light chain domain.

In various embodiments, the nucleic acid(s) (e.g., cDNA or genomic DNA)encoding the two heavy chains and single human light chain is insertedinto a replicable vector for further cloning (amplification of the DNA)and/or for expression. Many vectors are available, and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Each component may be selected individually or based on a host cellchoice or other criteria determined experimentally. Several examples ofeach component are known in the art.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid sequences that encode each or all the components of the bispecificantibody. A large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto bispecific antibody-encoding DNA by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) may also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the bispecific antibody components.Suitable expression vectors for various embodiments include those thatprovide for the transient expression in mammalian cells of DNA encodingthe bispecific antibody. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of bispecific antibodieshaving desired binding specificities/affinities or the desired gelmigration characteristics relative to the parental antibodies havinghomodimers of the first or second human heavy chain variable domains.

In various embodiments, once the DNA encoding the components of thebispecific antibody are assembled into the desired vector(s) asdescribed above, they are introduced into a suitable host cell forexpression and recovery. Transfecting host cells can be accomplishedusing standard techniques known in the art appropriate to the host cellselected (e.g., electroporation, nuclear microinjection, bacterialprotoplast fusion with intact cells, or polycations, e.g., polybrene,polyornithine, etc.).

A host cell is chosen, in various embodiments, that best suits theexpression vector containing the components and allows for the mostefficient and favorable production of the bispecific antibody species.Exemplary host cells for expression include those of prokaryotes andeukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In various embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In various embodiments,the cell is eukaryotic cell selected from CHO (e.g., CHO K1, DXB-11 CHO,Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5,Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal),CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertolicell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cellline derived from an aforementioned cell. In various embodiments, thecell comprises one or more viral genes, e.g. a retinal cell thatexpresses a viral gene (e.g., a PER.C6™ cell).

Mammalian host cells used to produce the bispecific antibody may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbeccols Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. Media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other supplements may also be includedat appropriate concentrations as known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are, invarious embodiments, those previously used with the host cell selectedfor expression, and will be apparent to those skilled in the art.

The bispecific antibody is in various embodiments recovered from theculture medium as a secreted polypeptide, although it also may berecovered from host cell lysate when directly produced without asecretory signal. If the bispecific antibody is membrane-bound, it canbe released from the membrane using a suitable detergent solution (e.g.,Triton-X 100). Preferably, the bispecific antibodies described hereininvolves the use of a first immunoglobulin C_(H)3 domain and a secondimmunoglobulin C_(H)3 domain, wherein the first and secondimmunoglobulin C_(H)3 domains differ from one another by at least oneamino acid, and wherein at least one amino acid difference reducesbinding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference (see U.S.2010/0331527A1; herein incorporated by reference). In one embodiment,the first immunoglobulin C_(H)3 domain binds Protein A and the secondimmunoglobulin C_(H)3 domain contains a mutation that reduces orabolishes Protein A binding such as an H95R modification (by IMGT exonnumbering; H435R by EU numbering). The second C_(H)3 may furthercomprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second C_(H)3 include: D16E,L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

Because of the dual nature of bispecific antibodies (i.e., may bespecific for different epitopes of one polypeptide or may containantigen-binding domains specific for more than one target polypeptide,see FIG. 7B; see also, e.g., Tutt et al., 1991, J. Immunol. 147:60-69;Kufer et al., 2004, Trends Biotechnol. 22:238-244), they offer manyuseful advantages for therapeutic application. For example, thebispecific antibodies can be used for redirected cytotoxicity (e.g., tokill tumor cells), as a vaccine adjuvant, for delivering thrombolyticagents to clots, for converting enzyme activated prodrugs at a targetsite (e.g., a tumor), for treating infectious diseases, targeting immunecomplexes to cell surface receptors, or for delivering immunotoxins totumor cells.

The bispecific antibodies described herein can also be used in severaltherapeutic and non-therapeutic and/or diagnostic assay methods, suchas, enzyme immunoassays, two-site immunoassays, in vitro or in vivoimmunodiagnosis of various diseases (e.g., cancer), competitive bindingassays, direct and indirect sandwich assays, and immunoprecipitationassays. Other uses for the bispecific antibodies will be apparent tothose skilled in the art.

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is indicated in Celsius, andpressure is at or near atmospheric.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, temperature is indicated in Celsius,pressure is at or near atmospheric, parts are by parts by weight, andmolecular weight is average molecular weight.

Example 1 Identification of Human V_(H) Regions that Associate withSelected Human V_(L) Regions

An in vitro expression system was constructed to determine if a singlerearranged human germline light chain could be co-expressed with humanheavy chains from antigen specific human antibodies.

Methods for generating human antibodies in genetically modified mice areknown (see e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals,VELOCIMMUNE®). The VELOCIMMUNE® technology involves generation of agenetically modified mouse having a genome comprising human heavy andlight chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibodies produced from a VELOCIMMUNE®mouse are fully human. Initially, high affinity chimeric antibodies areisolated having a human variable region and a mouse constant region. Asdescribed below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate a fully human antibody containing a non-IgMisotype, for example, wild type or modified IgG1, IgG2, IgG3 or IgG4.While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region.

A VELOCIMMUNE® mouse was immunized with a growth factor that promotesangiogenesis (Antigen C) and antigen-specific human antibodies wereisolated and sequenced for V gene usage using standard techniquesrecognized in the art. Selected antibodies were cloned onto human heavyand light chain constant regions and 69 heavy chains were selected forpairing with one of three human light chains: (1) the cognate κ lightchain linked to a human κ constant region, (2) a rearranged humangermline Vκ1-39Jκ5 linked to a human κ constant region, or (3) arearranged human germline Vκ3-20Jκ1 linked to a human κ constant region.Each heavy chain and light chain pair was co-transfected in CHO-K1 cellsusing standard techniques. Presence of antibody in the supernatant wasdetected by anti-human IgG in an ELISA assay. Antibody titer (ng/ml) wasdetermined for each heavy chain/light chain pair and titers with thedifferent rearranged germline light chains were compared to the titersobtained with the parental antibody molecule (i.e., heavy chain pairedwith cognate light chain) and percent of native titer was calculated(Table 1). V_(H): Heavy chain variable gene. ND: no expression detectedunder current experimental conditions.

TABLE 1 Antibody Titer (ng/mL) Percent of Native Titer Cognate Vκ1- Vκ3-Vκ1- Vκ3- V_(H) LC 39Jκ5 20Jκ1 39Jκ5 20Jκ1 3-15 63 23 11 36.2 17.5 1-2 103 53 ND 51.1 — 3-23 83 60 23 72.0 27.5 3-33 15 77 ND 499.4 — 4-31 2269 17 309.4 76.7 3-7  53 35 28 65.2 53.1 — 22 32 19 148.8 89.3 1-24 3 13ND 455.2 — 3-33 1 47 ND 5266.7 — 3-33 58 37 ND 63.1 — — 110 67 18 60.616.5 3-23 127 123 21 96.5 16.3 3-33 28 16 2 57.7 7.1 3-23 32 50 38 157.1119.4 — 18 45 18 254.3 101.7 3-9  1 30 23 2508.3 1900.0 3-11 12 26 6225.9 48.3 1-8  16 ND 13 — 81.8 3-33 54 81 10 150.7 19.1 — 34 9 ND 25.9— 3-20 7 14 54 203.0 809.0 3-33 19 38 ND 200.5 — 3-11 48 ND 203 — 423.6— 11 23 8 212.7 74.5 3-33 168 138 182 82.0 108.2 3-20 117 67 100 57.586.1 3-23 86 61 132 70.7 154.1 3-33 20 12 33 60.9 165.3 4-31 69 92 52133.8 75.0 3-23 87 78 62 89.5 71.2 1-2  31 82 51 263.0 164.6 3-23 53 93151 175.4 285.4 — 11 8 17 75.7 151.4 3-33 114 36 27 31.6 23.4 3-15 73 3944 53.7 59.6 3-33 1 34 16 5600.0 2683.3 3-9  58 112 57 192.9 97.6 3-3367 20 105 30.1 157.0 3-33 34 21 24 62.7 70.4 3-20 10 49 91 478.4 888.23-33 66 32 25 48.6 38.2 3-23 17 59 56 342.7 329.8 — 58 108 19 184.4 32.9— 68 54 20 79.4 29.9 3-33 42 35 32 83.3 75.4 — 29 19 13 67.1 43.9 3-9 24 34 29 137.3 118.4 3-30/33 17 33 7 195.2 43.1 3-7  25 70 74 284.6301.6 3-33 87 127 ND 145.1 — 6-1  28 56 ND 201.8 — 3-33 56 39 20 69.936.1 3-33 10 53 1 520.6 6.9 3-33 20 67 10 337.2 52.3 3-33 11 36 18 316.8158.4 3-23 12 42 32 356.8 272.9 3-33 66 95 15 143.6 22.5 3-15 55 68 ND123.1 — — 32 68 3 210.9 10.6 1-8  28 48 ND 170.9 — 3-33 124 192 21 154.317.0 3-33 0 113 ND 56550.0 — 3-33 10 157 1 1505.8 12.5 3-33 6 86 151385.5 243.5 3-23 70 115 22 163.5 31.0 3-7  71 117 21 164.6 29.6 3-33 82100 47 122.7 57.1 3-7  124 161 41 130.0 33.5

In a similar experiment, VELOCIMMUNE® mice were immunized with severaldifferent antigens and selected heavy chains of antigen specific humanantibodies were tested for their ability to pair with differentrearranged human germline light chains (as described above). Theantigens used in this experiment included an enzyme involved incholesterol homeostasis (Antigen A), a serum hormone involved inregulating glucose homeostasis (Antigen B), a growth factor thatpromotes angiogenesis (Antigen C) and a cell-surface receptor (AntigenD). Antigen specific antibodies were isolated from mice of eachimmunization group and the heavy chain and light chain variable regionswere cloned and sequenced. From the sequence of the heavy and lightchains, V gene usage was determined and selected heavy chains werepaired with either their cognate light chain or a rearranged humangermline Vκ1-39Jκ5 region. Each heavy/light chain pair wasco-transfected in CHO-K1 cells and the presence of antibody in thesupernatant was detected by anti-human IgG in an ELISA assay. Antibodytiter (μg/ml) was determined for each heavy chain/light chain pairingand titers with the different rearranged human germline light chainswere compared to the titers obtained with the parental antibody molecule(i.e., heavy chain paired with cognate light chain) and percent ofnative titer was calculated (Table 2). V_(H): Heavy chain variable gene.Vκ: κ light chain variable gene. ND: no expression detected undercurrent experimental conditions.

TABLE 2 Titer (μg/ml) V_(H) + Percent of Antigen Antibody V_(H) Vκ V_(H)Alone V_(H) + Vκ Vκ1-39Jκ5 Native Titer A 320 1-18 2-30 0.3 3.1 2.0 66321 2-5 2-28 0.4 0.4 1.9 448 334 2-5 2-28 0.4 2.7 2.0 73 313 3-13 3-150.5 0.7 4.5 670 316 3-23 4-1 0.3 0.2 4.1 2174 315 3-30 4-1 0.3 0.2 3.21327 318 4-59 1-17 0.3 4.6 4.0 86 B 257 3-13 1-5 0.4 3.1 3.2 104 2833-13 1-5 0.4 5.4 3.7 69 637 3-13 1-5 0.4 4.3 3.0 70 638 3-13 1-5 0.4 4.13.3 82 624 3-23 1-17 0.3 5.0 3.9 79 284 3-30 1-17 0.3 4.6 3.4 75 6533-33 1-17 0.3 4.3 0.3 7 268 4-34 1-27 0.3 5.5 3.8 69 633 4-34 1-27 0.66.9 3.0 44 C 730 3-7 1-5 0.3 1.1 2.8 249 728 3-7 1-5 0.3 2.0 3.2 157 6913-9 3-20 0.3 2.8 3.1 109 749 3-33 3-15 0.3 3.8 2.3 62 750 3-33 1-16 0.33.0 2.8 92 724 3-33 1-17 0.3 2.3 3.4 151 706 3-33 1-16 0.3 3.6 3.0 84744 1-18 1-12 0.4 5.1 3.0 59 696 3-11 1-16 0.4 3.0 2.9 97 685 3-13 3-200.3 0.5 3.4 734 732 3-15 1-17 0.3 4.5 3.2 72 694 3-15 1-5 0.4 5.2 2.9 55743 3-23 1-12 0.3 3.2 0.3 10 742 3-23 2-28 0.4 4.2 3.1 74 693 3-23 1-120.5 4.2 4.0 94 D 136 3-23 2-28 0.4 5.0 2.7 55 155 3-30 1-16 0.4 1.0 2.2221 163 3-30 1-16 0.3 0.6 3.0 506 171 3-30 1-16 0.3 1.0 2.8 295 145 3-431-5 0.4 4.4 2.9 65 49 3-48 3-11 0.3 1.7 2.6 155 51 3-48 1-39 0.1 1.9 0.14 159 3-7 6-21 0.4 3.9 3.6 92 169 3-7 6-21 0.3 1.3 3.1 235 134 3-9 1-50.4 5.0 2.9 58 141 4-31 1-33 2.4 4.2 2.6 63 142 4-31 1-33 0.4 4.2 2.8 67

The results obtained from these experiments demonstrate that somaticallymutated, high affinity heavy chains from different gene families areable to pair with rearranged human germline Vκ1-39Jκ5 and Vκ3-20Jκ1regions and be secreted from the cell as a normal antibody molecule. Asshown in Table 1, antibody titer was increased for about 61% (42 of 69)heavy chains when paired with the rearranged human Vκ1-39Jκ5 light chainand about 29% (20 of 69) heavy chains when paired with the rearrangedhuman Vκ3-20Jκ1 light chain as compared to the cognate light chain ofthe parental antibody. For about 20% (14 of 69) of the heavy chains,both rearranged human germline light chains conferred an increase inexpression as compared to the cognate light chain of the parentalantibody. As shown in Table 2, the rearranged human germline Vκ1-39Jκ5region conferred an increase in expression of several heavy chainsspecific for a range of different classes of antigens as compared to thecognate light chain for the parental antibodies. Antibody titer wasincreased by more than two-fold for about 35% (15/43) of the heavychains as compared to the cognate light chain of the parentalantibodies. For two heavy chains (315 and 316), the increase was greaterthan ten-fold as compared to the parental antibody. Within all the heavychains that showed increase expression relative to the cognate lightchain of the parental antibody, family three (V_(H)3) heavy chains areover represented in comparison to other heavy chain variable region genefamilies. This demonstrates a favorable relationship of human V_(H)3heavy chains to pair with rearranged human germline Vκ1-39Jκ5 andVκ3-20Jκ1 light chains.

Example 2 Generation of a Rearranged Human Germline Light Chain Locus

Various rearranged human germline light chain targeting vectors weremade using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251and Valenzuela et al. (2003) High-throughput engineering of the mousegenome coupled with high-resolution expression analysis, Nature Biotech.21(6): 652-659) to modify mouse genomic Bacterial Artificial Chromosome(BAC) clones 302g12 and 254 m04 (Invitrogen). Using these two BACclones, genomic constructs were engineered to contain a singlerearranged human germline light chain region and inserted into anendogenous κ light chain locus that was previously modified to deletethe endogenous κ variable and joining gene segments.

Construction of Rearranged Human Germline Light Chain Targeting Vectors.

Three different rearranged human germline light chain regions were madeusing standard molecular biology techniques recognized in the art. Thehuman variable gene segments used for constructing these three regionsincluded rearranged human Vκ1-39Jκ5 sequence, a rearranged humanVκ3-20Jκ1 sequence and a rearranged human VpreBJλ5 sequence.

A DNA segment containing exon 1 (encoding the leader peptide) and intron1 of the mouse Vκ3-7 gene was made by de novo DNA synthesis (IntegratedDNA Technologies). Part of the 5′ untranslated region up to a naturallyoccurring Blpl restriction enzyme site was included. Exons of humanVκ1-39 and Vκ3-20 genes were PCR amplified from human genomic BAClibraries. The forward primers had a 5′ extension containing the spliceacceptor site of intron 1 of the mouse Vκ3-7 gene. The reverse primerused for PCR of the human Vκ1-39 sequence included an extension encodinghuman Jκ5, whereas the reverse primer used for PCR of the human Vκ3-20sequence included an extension encoding human JO. The human VpreBJλ5sequence was made by de novo DNA synthesis (Integrated DNATechnologies). A portion of the human Jκ-Cκ intron including the splicedonor site was PCR amplified from plasmid pBS-296-HA18-PISceI. Theforward PCR primer included an extension encoding part of either a humanJκ5, Jκ1, or Jλ5 sequence. The reverse primer included a PI-SceI site,which was previously engineered into the intron.

The mouse Vκ3-7 exon1/intron 1, human variable light chain exons, andhuman Jκ-Cκ intron fragments were sewn together by overlap extensionPCR, digested with Blpl and PI-SceI, and ligated into plasmidpBS-296-HA18-PISceI, which contained the promoter from the human Vκ3-15variable gene segment. A loxed hygromycin cassette within plasmidpBS-296-HA18-PISceI was replaced with a FRTed hygromycin cassetteflanked by NotI and AscI sites. The NotI/PI-SceI fragment of thisplasmid was ligated into modified mouse BAC 254 m04, which containedpart of the mouse Jκ-Cκ intron, the mouse Cκ exon, and about 75 kb ofgenomic sequence downstream of the mouse κ locus, which provided a 3′homology arm for homologous recombination in mouse ES cells. TheNotI/AscI fragment of this BAC was then ligated into modified mouse BAC302g12, which contained a FRTed neomycin cassette and about 23 kb ofgenomic sequence upstream of the endogenous κ locus for homologousrecombination in mouse ES cells.

Rearranged Human Germline Vκ1-39Jκ5 Targeting Vector (FIG. 1).

Restriction enzyme sites were introduced at the 5′ and 3′ ends of anengineered light chain insert for cloning into a targeting vector: anAscI site at the 5′ end and a PI-SceI site at the 3′ end. Within the 5′AscI site and the 3′ PI-SceI site the targeting construct from 5′ to 3′included a 5′ homology arm containing sequence 5′ to the endogenousmouse κ light chain locus obtained from mouse BAC clone 302g12, a FRTedneomycin resistance gene, an genomic sequence including the human Vκ3-15promoter, a leader sequence of the mouse Vκ3-7 variable gene segment, aintron sequence of the mouse Vκ3-7 variable gene segment, an openreading frame of a rearranged human germline Vκ1-39Jκ5 region, a genomicsequence containing a portion of the human Jκ-Cκ intron, and a 3′homology arm containing sequence 3′ of the endogenous mouse Jκ5 genesegment obtained from mouse BAC clone 254 m04 (FIG. 1, middle). Genesand/or sequences upstream of the endogenous mouse κ light chain locusand downstream of the most 3′ Jκ gene segment (e.g., the endogenous 3′enhancer) were unmodified by the targeting construct (see FIG. 1). Thesequence of the engineered human Vκ1-39Jκ5 locus is shown in SEQ ID NO:1.

Targeted insertion of the rearranged human germline Vκ1-39Jκ5 regioninto BAC DNA was confirmed by polymerase chain reaction (PCR) usingprimers located at sequences within the rearranged human germline lightchain region. Briefly, the intron sequence 3′ to the mouse Vκ3-7 leadersequence was confirmed with primers ULC-m1F (AGGTGAGGGT ACAGATAAGTGTTATGAG; SEQ ID NO: 2) and ULC-m1R (TGACAAATGC CCTAATTATA GTGATCA; SEQID NO: 3). The open reading frame of the rearranged human germlineVκ1-39Jκ5 region was confirmed with primers 1633-h2F (GGGCAAGTCAGAGCATTAGC A; SEQ ID NO: 4) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQID NO: 5). The neomycin cassette was confirmed with primers neoF(GGTGGAGAGG CTATTCGGC; SEQ ID NO: 6) and neoR (GAACACGGCG GCATCAG; SEQID NO: 7). Targeted BAC DNA was then used to electroporate mouse EScells to created modified ES cells for generating chimeric mice thatexpress a rearranged human germline Vκ1-39Jκ5 region.

Positive ES cell clones were confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered Vκ1-39Jκ5 lightchain region inserted into the endogenous locus. Briefly, probe neoP(TGGGCACAAC AGACAATCGG CTG; SEQ ID NO: 8) which binds within theneomycin marker gene, probe ULC-m1P (CCATTATGAT GCTCCATGCC TCTCTGTTC;SEQ ID NO: 9) which binds within the intron sequence 3′ to the mouseVκ3-7 leader sequence, and probe 1633h2P (ATCAGCAGAA ACCAGGGAAA GCCCCT;SEQ ID NO: 10) which binds within the rearranged human germlineVκ1-39Jκ5 open reading frame. Positive ES cell clones were then used toimplant female mice to give rise to a litter of pups expressing thegermline Vκ1-39Jκ5 light chain region.

Alternatively, ES cells bearing the rearranged human germline Vκ1-39Jκ5light chain region are transfected with a construct that expresses FLPin order to remove the FRTed neomycin cassette introduced by thetargeting construct. Optionally, the neomycin cassette is removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Rearranged Human Germline Vκ3-20Jκ1 Targeting Vector (FIG. 2).

In a similar fashion, an engineered light chain locus expressing arearranged human germline Vκ3-20Jκ1 region was made using a targetingconstruct including, from 5′ to 3′, a 5′ homology arm containingsequence 5′ to the endogenous mouse κ light chain locus obtained frommouse BAC clone 302g12, a FRTed neomycin resistance gene, a genomicsequence including the human Vκ3-15 promoter, a leader sequence of themouse Vκ3-7 variable gene segment, an intron sequence of the mouse Vκ3-7variable gene segment, an open reading frame of a rearranged humangermline Vκ3-20Jκ1 region, a genomic sequence containing a portion ofthe human Jκ-Cκ intron, and a 3′ homology arm containing sequence 3′ ofthe endogenous mouse Jκ5 gene segment obtained from mouse BAC clone 254m04 (FIG. 2, middle). The sequence of the engineered human Vκ3-20Jκ1locus is shown in SEQ ID NO: 11.

Targeted insertion of the rearranged human germline Vκ3-20Jκ1 regioninto BAC DNA was confirmed by polymerase chain reaction (PCR) usingprimers located at sequences within the rearranged human germlineVκ3-20Jκ1 light chain region. Briefly, the intron sequence 3′ to themouse Vκ3-7 leader sequence was confirmed with primers ULC-m1F (SEQ IDNO: 2) and ULC-m1R (SEQ ID NO: 3). The open reading frame of therearranged human germline Vκ3-20Jκ1 region was confirmed with primers1635-h2F (TCCAGGCACC CTGTCTTTG; SEQ ID NO: 12) and 1635-h2R (AAGTAGCTGCTGCTAACACT CTGACT; SEQ ID NO: 13). The neomycin cassette was confirmedwith primers neoF (SEQ ID NO: 6) and neoR (SEQ ID NO: 7). Targeted BACDNA was then used to electroporate mouse ES cells to created modified EScells for generating chimeric mice that express the rearranged humangermline Vκ3-20Jκ1 light chain.

Positive ES cell clones were confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered Vκ3-20Jκ1 lightchain region inserted into the endogenous κ light chain locus. Briefly,probe neoP (SEQ ID NO: 8) which binds within the neomycin marker gene,probe ULC-m1P (SEQ ID NO: 9) which binds within the mouse Vκ3-7 leadersequence, and probe 1635h2P (AAAGAGCCAC CCTCTCCTGC AGGG; SEQ ID NO: 14)which binds within the human Vκ3-20Jκ1 open reading frame. Positive EScell clones were then used to implant female mice. A litter of pupsexpressing the human germline Vκ3-20Jκ1 light chain region.

Alternatively, ES cells bearing human germline Vκ3-20Jκ1 light chainregion can be transfected with a construct that expresses FLP in orderto remove the FRTed neomycin cassette introduced by the targetingconstruct. Optionally, the neomycin cassette may be removed by breedingto mice that express FLP recombinase (e.g., U.S. Pat. No. 6,774,279).Optionally, the neomycin cassette is retained in the mice.

Rearranged Human Germline VpreBJλ5 Targeting Vector (FIG. 3).

In a similar fashion, an engineered light chain locus expressing arearranged human germline VpreBJλ5 region was made using a targetingconstruct including, from 5′ to 3′, a 5′ homology arm containingsequence 5′ to the endogenous mouse κ light chain locus obtained frommouse BAC clone 302g12, a FRTed neomycin resistance gene, an genomicsequence including the human Vκ3-15 promoter, a leader sequence of themouse Vκ3-7 variable gene segment, an intron sequence of the mouse Vκ3-7variable gene segment, an open reading frame of a rearranged humangermline VpreBJλ5 region, a genomic sequence containing a portion of thehuman Jκ-Cκ intron, and a 3′ homology arm containing sequence 3′ of theendogenous mouse Jκ5 gene segment obtained from mouse BAC clone 254 m04(FIG. 3, middle). The sequence of the engineered human VpreBJλ5 locus isshown in SEQ ID NO: 15.

Targeted insertion of the rearranged human germline VpreBJλ5 region intoBAC DNA was confirmed by polymerase chain reaction (PCR) using primerslocated at sequences within the rearranged human germline VpreBJλ5region light chain region. Briefly, the intron sequence 3′ to the mouseVκ3-7 leader sequence was confirmed with primers ULC-m1F (SEQ ID NO: 2)and ULC-m1R (SEQ ID NO: 3). The open reading frame of the rearrangedhuman germline VpreBJλ5 region was confirmed with primers 1616-h1F(TGTCCTCGGC CCTTGGA; SEQ ID NO: 16) and 1616-h1R(CCGATGTCAT GGTCGTTCCT;SEQ ID NO: 17). The neomycin cassette was confirmed with primers neoF(SEQ ID NO: 6) and neoR (SEQ ID NO: 7). Targeted BAC DNA was then usedto electroporate mouse ES cells to created modified ES cells forgenerating chimeric mice that express the rearranged human germlineVpreBJλ5 light chain.

Positive ES cell clones are confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered VpreBJλ5 lightchain region inserted into the endogenous κ light chain locus. Briefly,probe neoP (SEQ ID NO:8), which binds within the neomycin marker gene,probe ULC-m1P (SEQ ID NO: 9), which binds within the mouse IgVκ3-7leader sequence, and probe 1616h1P (ACAATCCGCC TCACCTGCAC CCT; SEQ IDNO: 18) which binds within the human VpreBJλ5 open reading frame.Positive ES cell clones are then used to implant female mice to giverise to a litter of pups expressing a germline light chain region.

Alternatively, ES cells bearing the rearranged human germline VpreBJλ5light chain region are transfected with a construct that expresses FLPin order to remove the FRTed neomycin cassette introduced by thetargeting construct. Optionally, the neomycin cassette is removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Example 3 Generation of Mice Expressing a Single Rearranged Human LightChain

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) FOgeneration mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1): 91-99. VELOCIMICE® independently bearing an engineeredhuman germline Vκ1-39Jκ5 light chain region, a Vκ3-20Jκ1 light chainregion or a VpreBJλ5 light chain region are identified by genotypingusing a modification of allele assay (Valenzuela et al., supra) thatdetects the presence of the unique rearranged human germline light chainregion.

Pups are genotyped and a pup heterozygous or homozygous for the uniquerearranged human germline light chain region are selected forcharacterizing expression of the rearranged human germline light chainregion.

Flow Cytometry.

Expression of the rearranged human light chain region in the normalantibody repertoire of common light chain mice was validated by analysisof immunoglobulin κ and λ expression in splenocytes and peripheral bloodof common light chain mice. Cell suspensions from harvested spleens andperipheral blood of wild type (n=5), Vκ1-39Jκ5 common light chainheterozygote (n=3), Vκ1-39Jκ5 common light chain homozygote (n=3),Vκ3-20Jκ1 common light chain heterozygote (n=2), and Vκ3-20Jκ1 commonlight chain homozygote (n=2) mice were made using standard methods andstained with CD19⁺, Igλ⁺ and Igκ⁺ using fluorescently labeled antibodies(BD Pharmigen).

Briefly, 1×10⁶ cells were incubated with anti-mouse CD16/CD32 (clone2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling withthe following antibody cocktail for 30 minutes on ice: APC conjugatedanti-mouse CD19 (clone 1D3, BD Pharmigen), PerCP-Cy5.5 conjugatedanti-mouse CD3 (clone 17A2, BioLegend), FITC conjugated anti-mouse Igκ(clone 187.1, BD Pharmigen), PE conjugated anti-mouse Igλ (clone RML-42,BioLegend). Following staining, cells were washed and fixed in 2%formaldehyde. Data acquisition was performed on an LSRII flow cytometerand analyzed with FlowJo. Gating: total B cells (CD19⁺CD3⁻), Igκ⁺ Bcells (Igκ⁺Igλ⁻CD19⁺CD3⁻), Igλ⁺ B cells (Igκ⁺Igλ⁺CD19⁺CD3⁻). Datagathered from blood and splenocyte samples demonstrated similar results.Table 3 sets forth the percent positive CD19⁺ B cells from peripheralblood of one representative mouse from each group that are Igλ⁺, Igκ⁺,or Igλ⁺Igκ⁺. Percent of CD19⁺ B cells in peripheral blood from wild type(WT) and mice homozygous for either the Vκ1-39Jκ5 or Vκ3-20Jκ1 commonlight chain are shown in FIG. 4.

TABLE 3 CD19⁺ B Cells Mouse Igλ⁺ Igκ⁺ Igλ⁺Igκ⁺ Wild type 4.8 93 0.53Vκ1-39Jκ5 1.4 93 2.6 Vκ3-20Jκ1 4.2 88 6

Common Light Chain Expression.

Expression of each common light chain (Vκ1-39Jκ5 and Vκ3-20Jκ1) wasanalyzed in heterozygous and homozygous mice using a quantitative PCRassay (e.g. TAQMAN™).

Briefly, CD19⁺ B cells were purified from the spleens of wild type, micehomozygous for a replacement of the mouse heavy chain and κ light chainvariable region loci with corresponding human heavy chain and κ lightchain variable region loci (Hκ), as well as mice homozygous andheterozygous for each rearranged human light chain region (Vκ1-39Jκ5 orVκ3-20Jκ1) using mouse CD19 Microbeads (Miltenyi Biotec) according tomanufacturer's specifications. Total RNA was purified from CD19⁺ B cellsusing RNeasy Mini kit (Qiagen) according to manufacturer'sspecifications and genomic RNA was removed using an RNase-free DNaseon-column treatment (Qiagen). 200 ng mRNA was reverse-transcribed intocDNA using the First Stand cDNA Synthesis kit (Invitrogen) and theresulting cDNA was amplified with the Taqman Universal PCR Master Mix(Applied Biosystems). All reactions were performed using the ABI 7900Sequence Detection System (Applied Biosystems) using primers and TaqmanMGB probes spanning (1) the Vκ-Jκ junction for both common light chains,(2) the Vκ gene alone (i.e. Vκ1-39 and Vκ3-20), and (3) the mouse Cκregion. Table 4 sets forth the sequences of the primers and probesemployed for this assay. Relative expression was normalized toexpression of the mouse Cκ region. Results are shown in FIGS. 5A, 5B and5C.

TABLE 4 SEQ Primer/Probe Description ID Region (5′-3′) NOs: Vκ1-39Jκ5(Sense)  19 Junction AGCAGTCTGC AACCTGAAGA TTT (Anti-sense)  20GTTTAATCTC CAGTCGTGTC CCTT (Probe)  21 CCTCCGATCA CCTTC Vκ1-39 (Sense) 22 AAACCAGGGA AAGCCCCTAA (Anti-sense)  23 ATGGGACCCC ACTTTGCA (Probe) 24 CTCCTGATCT ATGCTGCAT Vκ3-20Jκ1 (Sense)  25 JunctionCAGCAGACTG GAGCCTGAAG A (Anti-sense)  26 TGATTTCCAC CTTGGTCCCT T(Probe)  27 TAGCTCACCT TGGACGTT Vκ3-20 (Sense)  28CTCCTCATCT ATGGTGCATC CA (Anti-sense)  29 GACCCACTGC CACTGAACCT (Probe) 30 CCACTGGCAT CCC Mouse Cκ (Sense)  31 TGAGCAGCAC CCTCACGTT(Anti-sense)  32 GTGGCCTCAC AGGTATAGCT GTT (Probe)  33ACCAAGGACG AGTATGAA

Antigen Specific Common Light Chain Antibodies.

Common light chain mice bearing either a Vκ1-39Jκ5 or Vκ3-20Jκ1 commonlight chain at the endogenous mouse κ light chain locus were immunizedwith β-galactosidase and antibody titer was measured.

Briefly, β-galactosidase (Sigma) was emulsified in titermax adjuvant(Sigma), as per manufacturer's directions. Wild type (n=7), Vκ1-39Jκ5common light chain homozygotes (n=2) and Vκ3-20Jκ1 common light chainhomozygotes (n=5) were immunized by subcutaneous injection with 100 μgβ-galactosidase/Titermax. Mice were boosted by subcutaneous injectiontwo times, 3 weeks apart, with 50 μg β-galactosidase/Titermax. After thesecond boost, blood was collected from anaesthetized mice using aretro-orbital bleed into serum separator tubes (BD Biosciences) as permanufacturer's directions. To measure anti-β-galactosidase IgM or IgGantibodies, ELISA plates (Nunc) were coated with 1 μg/mL β-galactosidaseovernight at 4° C. Excess antigen was washed off before blocking withPBS with 1% BSA for one hour at room temperature. Serial dilutions ofserum were added to the plates and incubated for one hour at roomtemperature before washing. Plates were then incubated with HRPconjugated anti-IgM (Southern Biotech) or anti-IgG (Southern Biotech)for one hour at room temperature. Following another wash, plates weredeveloped with TMB substrate (BD Biosciences). Reactions were stoppedwith 1N sulfuric acid and OD₄₅₀ was read using a Victor X5 Plate Reader(Perkin Elmer). Data was analyzed with GraphPad Prism and signal wascalculated as the dilution of serum that is two times above background.Results are shown in FIGS. 6A and 6B.

As shown in this Example, the ratio of κ/λ B cells in both the splenicand peripheral compartments of Vκ1-39Jκ5 and Vκ3-20Jκ1 common lightchain mice demonstrated a near wild type pattern (Table 3 and FIG. 4).VpreBJλ5 common light chain mice, however, demonstrated fewer peripheralB cells, of which about 1-2% express the engineered human light chainregion (data not shown). The expression levels of the Vκ1-39Jκ5 andVκ3-20Jκ1 rearranged human light chain regions from the endogenous κlight chain locus were elevated in comparison to an endogenous κ lightchain locus containing a complete replacement of mouse Vκ and Jκ genesegments with human Vκ and Jκ gene segments (FIGS. 5A, 5B and 5C). Theexpression levels of the VpreBJλ5 rearranged human light chain regiondemonstrated similar high expression from the endogenous κ light chainlocus in both heterozygous and homozygous mice (data not shown). Thisdemonstrates that in direct competition with the mouse 2, K, or bothendogenous light chain loci, a single rearranged human V_(L)/J_(L)sequence can yield better than wild type level expression from theendogenous κ light chain locus and give rise to normal splenic and bloodB cell frequency. Further, the presence of an engineered κ light chainlocus having either a human Vκ1-39Jκ5 or human Vκ3-20Jκ1 sequence waswell tolerated by the mice and appear to function in wild type fashionby representing a substantial portion of the light chain repertoire inthe humoral component of the immune response (FIGS. 6A and 6B).

Example 4 Breeding of Mice Expressing a Single Rearranged Human GermlineLight Chain

This Example describes several other genetically modified mouse strainsthat can be bred to any one of the common light chain mice describedherein to create multiple genetically modified mouse strains harboringmultiple genetically modified immunoglobulin loci.

Endogenous Igλ Knockout (KO).

To optimize the usage of the engineered light chain locus, mice bearingone of the rearranged human germline light chain regions are bred toanother mouse containing a deletion in the endogenous λ light chainlocus. In this manner, the progeny obtained will express, as their onlylight chain, the rearranged human germline light chain region asdescribed in Example 2. Breeding is performed by standard techniquesrecognized in the art and, alternatively, by a commercial breeder (e.g.,The Jackson Laboratory). Mouse strains bearing an engineered light chainlocus and a deletion of the endogenous λ light chain locus are screenedfor presence of the unique light chain region and absence of endogenousmouse λ light chains.

Humanized Endogenous Heavy Chain Locus.

Mice bearing an engineered human germline light chain locus are bredwith mice that contain a replacement of the endogenous mouse heavy chainvariable gene locus with the human heavy chain variable gene locus (seeU.S. Pat. No. 6,596,541; the VELOCIMMUNE® mouse, RegeneronPharmaceuticals, Inc.). The VELOCIMMUNE® mouse comprises a genomecomprising human heavy chain variable regions operably linked toendogenous mouse constant region loci such that the mouse producesantibodies comprising a human heavy chain variable region and a mouseheavy chain constant region in response to antigenic stimulation. TheDNA encoding the variable regions of the heavy chains of the antibodiesis isolated and operably linked to DNA encoding the human heavy chainconstant regions. The DNA is then expressed in a cell capable ofexpressing the fully human heavy chain of the antibody.

Mice bearing a replacement of the endogenous mouse V_(H) locus with thehuman VH locus and a single rearranged human germline V_(L) region atthe endogenous κ light chain locus are obtained. Reverse chimericantibodies containing somatically mutated heavy chains (human V_(H) andmouse C_(H)) with a single human light chain (human V_(L) and mouseC_(L)) are obtained upon immunization with an antigen of interest. V_(H)and V_(L) nucleotide sequences of B cells expressing the antibodies areidentified and fully human antibodies are made by fusion the V_(H) andV_(L) nucleotide sequences to human C_(H) and C_(L) nucleotide sequencesin a suitable expression system.

Example 5 Generation of Antibodies from Mice Expressing Human HeavyChains and a Rearranged Human Germline Light Chain Region

After breeding mice that contain the engineered human light chain regionto various desired strains containing modifications and deletions ofother endogenous Ig loci (as described in Example 4), selected mice canbe immunized with an antigen of interest.

Generally, a VELOCIMMUNE® mouse containing one of the single rearrangedhuman germline light chain regions is challenged with an antigen, andlymphatic cells (such as B-cells) are recovered from serum of theanimals. The lymphatic cells are fused with a myeloma cell line toprepare immortal hybridoma cell lines, and such hybridoma cell lines arescreened and selected to identify hybridoma cell lines that produceantibodies containing human heavy chain variables and a rearranged humangermline light chains which are specific to the antigen used forimmunization. DNA encoding the variable regions of the heavy chains andthe light chain are isolated and linked to desirable isotypic constantregions of the heavy chain and light chain. Due to the presence of theendogenous mouse sequences and any additional cis-acting elementspresent in the endogenous locus, the single light chain of each antibodymay be somatically mutated. This adds additional diversity to theantigen-specific repertoire comprising a single light chain and diverseheavy chain sequences. The resulting cloned antibody sequences aresubsequently expressed in a cell, such as a CHO cell. Alternatively, DNAencoding the antigen-specific chimeric antibodies or the variabledomains of the light and heavy chains is identified directly fromantigen-specific lymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As described above, theantibodies are characterized and selected for desirable characteristics,including affinity, selectivity, epitope, etc. The mouse constantregions are replaced with a desired human constant region to generatethe fully human antibody containing a somatically mutated human heavychain and a single light chain derived from a rearranged human germlinelight chain region of the invention. Suitable human constant regionsinclude, for example wild type or modified IgG1 or IgG4.

Separate cohorts of VELOCIMMUNE® mice containing a replacement of theendogenous mouse heavy chain locus with human V_(H), D_(H), and J_(H)gene segments and a replacement of the endogenous mouse κ light chainlocus with either the engineered germline Vκ1-39Jκ5 human light chainregion or the engineered germline Vκ3-20Jκ1 human light chain region(described above) were immunized with a human cell-surface receptorprotein (Antigen E). Antigen E is administered directly onto the hindfootpad of mice with six consecutive injections every 3-4 days. Two tothree micrograms of Antigen E are mixed with 10 μg of CpGoligonucleotide (Cat # tlrl-modn—ODN1826 oligonucleotide; InVivogen, SanDiego, Calif.) and 25 μg of Adju-Phos (Aluminum phosphate gel adjuvant,Cat # H-71639-250; Brenntag Biosector, Frederikssund, Denmark) prior toinjection. A total of six injections are given prior to the finalantigen recall, which is given 3-5 days prior to sacrifice. Bleeds afterthe 4th and 6th injection are collected and the antibody immune responseis monitored by a standard antigen-specific immunoassay.

When a desired immune response is achieved splenocytes are harvested andfused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines are screened and selectedto identify cell lines that produce Antigen E-specific common lightchain antibodies. Using this technique several anti-Antigen E-specificcommon light chain antibodies (i.e., antibodies possessing human heavychain variable domains, the same human light chain variable domain, andmouse constant domains) are obtained.

Alternatively, anti-Antigen E common light chain antibodies are isolateddirectly from antigen-positive B cells without fusion to myeloma cells,as described in U.S. 2007/0280945A1, herein specifically incorporated byreference in its entirety. Using this method, several fully humananti-Antigen E common light chain antibodies (i.e., antibodiespossessing human heavy chain variable domains, either an engineeredhuman Vκ1-39Jκ5 light chain or an engineered human Vκ3-20Jκ1 light chainregion, and human constant domains) were obtained.

The biological properties of the exemplary anti-Antigen E common lightchain antibodies generated in accordance with the methods of thisExample are described in detail in the sections set forth below.

Example 6 Heavy Chain Gene Segment Usage in Antigen-Specific CommonLight Chain Antibodies

To analyze the structure of the human anti-Antigen E common light chainantibodies produced, nucleic acids encoding heavy chain antibodyvariable regions were cloned and sequenced. From the nucleic acidsequences and predicted amino acid sequences of the antibodies, geneusage was identified for the heavy chain variable region (HCVR) ofselected common light chain antibodies obtained from immunizedVELOCIMMUNE® mice containing either the engineered human Vκ1-39Jκ5 lightchain or engineered human Vκ3-20Jκ1 light chain region. Results areshown in Tables 5 and 6, which demonstrate that mice according to theinvention generate antigen-specific common light chain antibodies from avariety of human heavy chain gene segments, due to a variety ofrearrangements, when employing either a mouse that expresses a lightchain from only a human Vκ1-39- or a human Vκ3-20-derived light chain.Human V_(H) gene segments of the 2, 3, 4, and 5 families rearranged witha variety of human D_(H) segments and human J_(H) segments to yieldantigen-specific antibodies.

TABLE 5 Vκ1-39Jκ5 Common Light Chain Antibodies HCVR Antibody V_(H)D_(H) J_(H) 2952 2-5 6-6 1 5978 2-5 6-6 1 5981 2-5 3-22 1 6027 3-13 6-65 3022 3-23 3-10 4 3028 3-23 3-3 4 5999 3-23 6-6 4 6009 3-23 2-8 4 60113-23 7-27 4 5980 3-30 1-1 4 3014 3-30 1-7 4 3015 3-30 1-7 4 3023 3-301-7 4 3024 3-30 1-7 4 3032 3-30 1-7 4 6024 3-30 1-7 4 6025 3-30 1-7 46031 3-30 1-7 4 6007 3-30 3-3 4 2982 3-30 3-22 5 6001 3-30 3-22 5 60053-30 3-22 5 6035 3-30 5-5 2 3013 3-30 5-12 4 3042 3-30 5-12 4 2955 3-306-6 1 3043 3-30 6-6 3 3018 3-30 6-6 4 2949 3-30 6-6 5 2950 3-30 6-6 52954 3-30 6-6 5 2978 3-30 6-6 5 3016 3-30 6-6 5 3017 3-30 6-6 5 30333-30 6-6 5 3041 3-30 6-6 5 5979 3-30 6-6 5 5998 3-30 6-6 5 6004 3-30 6-65 6010 3-30 6-6 5 6019 3-30 6-6 5 6021 3-30 6-6 5 6022 3-30 6-6 5 60233-30 6-6 5 6030 3-30 6-6 5 6032 3-30 6-6 5 2985 3-30 6-13 4 2997 3-306-13 4 3011 3-30 6-13 4 3047 3-30 6-13 4 5982 3-30 6-13 4 6002 3-30 6-134 6003 3-30 6-13 4 6012 3-30 6-13 4 6013 3-30 6-13 4 6014 3-30 6-13 46015 3-30 6-13 4 6016 3-30 6-13 4 6017 3-30 6-13 4 6020 3-30 6-13 4 60343-30 6-13 4 2948 3-30 7-27 4 2987 3-30 7-27 4 2996 3-30 7-27 4 3005 3-307-27 4 3012 3-30 7-27 4 3020 3-30 7-27 4 3021 3-30 7-27 4 3025 3-30 7-274 3030 3-30 7-27 4 3036 3-30 7-27 4 5997 3-30 7-27 4 6033 3-30 7-27 43004 3-30 7-27 5 6028 3-30 7-27 6 3010 4-59 3-16 3 3019 4-59 3-16 3 60184-59 3-16 3 6026 4-59 3-16 3 6029 4-59 3-16 3 6036 4-59 3-16 3 6037 4-593-16 3 2964 4-59 3-22 3 3027 4-59 3-16 4 3046 5-51 5-5 3 6000 1-69 6-134 6006 1-69 6-6 5 6008 1-69 6-13 4

TABLE 6 Vκ3-20Jκ1 Common Light Chain Antibodies HCVR Antibody V_(H)D_(H) J_(H) 5989 3-30 3-3 3 5994 3-33 1-7 4 5985 3-33 2-15 4 5987 3-332-15 4 5995 3-33 2-15 4 2968 4-39 1-26 3 5988 4-39 1-26 3 5990 4-39 1-263 5992 4-39 1-26 3 2975 5-51 6-13 5 2972 5-51 3-16 6 5986 5-51 3-16 65993 5-51 3-16 6 5996 5-51 3-16 6 5984 3-53 1-1 4

Example 7 Determination of Blocking Ability of Antigen-Specific CommonLight Chain Antibodies by LUMINEX™ Assay

Ninety-eight human common light chain antibodies raised against AntigenE were tested for their ability to block binding of Antigen E's naturalligand (Ligand Y) to Antigen E in a bead-based assay.

The extracellular domain (ECD) of Antigen E was conjugated to two mycepitope tags and a 6× histidine tag (Antigen E-mmH) and amine-coupled tocarboxylated microspheres at a concentration of 20 μg/mL in MES buffer.The mixture was incubated for two hours at room temperature followed bybead deactivation with 1M Tris pH 8.0 followed by washing in PBS with0.05% (v/v) Tween-20. The beads were then blocked with PBS (IrvineScientific, Santa Ana, Calif.) containing 2% (w/v) BSA (Sigma-AldrichCorp., St. Louis, Mo.). In a 96-well filter plate, supernatantscontaining Antigen E-specific common light chain antibodies were diluted1:15 in buffer. A negative control containing a mock supernatant withthe same media components as for the antibody supernatant was prepared.Antigen E-labeled beads were added to the supernatants and incubatedovernight at 4° C. Biotinylated-Ligand Y protein was added to a finalconcentration of 0.06 nM and incubated for two hours at roomtemperature. Detection of biotinylated-Ligand Y bound to AntigenE-myc-myc-6His labeled beads was determined with R-Phycoerythrinconjugated to Streptavidin (Moss Inc, Pasadena, Md.) followed bymeasurement in a LUMINEX™ flow cytometry-based analyzer. Background MeanFluorescence Intensity (MFI) of a sample without Ligand Y was subtractedfrom all samples. Percent blocking was calculated by division of thebackground-subtracted MFI of each sample by the adjusted negativecontrol value, multiplying by 100 and subtracting the resulting valuefrom 100.

In a similar experiment, the same 98 human common light chain antibodiesraised against Antigen E were tested for their ability to block bindingof Antigen E to Ligand Y-labeled beads.

Briefly, Ligand Y was amine-coupled to carboxylated microspheres at aconcentration of 20 μg/mL diluted in MES buffer. The mixture andincubated two hours at room temperature followed by deactivation ofbeads with 1M Tris pH 8 then washing in PBS with 0.05% (v/v) Tween-20.The beads were then blocked with PBS (Irvine Scientific, Santa Ana,Calif.) containing 2% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, Mo.).In a 96-well filter plate, supernatants containing Antigen E-specificcommon light chain antibodies were diluted 1:15 in buffer. A negativecontrol containing a mock supernatant with the same media components asfor the antibody supernatant was prepared. A biotinylated-Antigen E-mmHwas added to a final concentration of 0.42 nM and incubated overnight at4° C. Ligand Y-labeled beads were then added to the antibody/Antigen Emixture and incubated for two hours at room temperature. Detection ofbiotinylated-Antigen E-mmH bound to Ligand Y-beads was determined withR-Phycoerythrin conjugated to Streptavidin (Moss Inc, Pasadena, Md.)followed by measurement in a LUMINEX™ flow cytometry-based analyzer.Background Mean Fluorescence Intensity (MFI) of a sample without AntigenE was subtracted from all samples. Percent blocking was calculated bydivision of the background-subtracted MFI of each sample by the adjustednegative control value, multiplying by 100 and subtracting the resultingvalue from 100.

Tables 7 and 8 show the percent blocking for all 98 anti-Antigen Ecommon light chain antibodies tested in both LUMINEX™ assays. ND: notdetermined under current experimental conditions.

TABLE 7 Vκ1-39Jκ5 Common Light Chain Antibodies % Blocking of % Blockingof Antibody Antigen E-Labeled Beads Antigen E In Solution 2948 81.1 47.82948G 38.6 ND 2949 97.6 78.8 2949G 97.1 73.7 2950 96.2 81.9 2950G 89.831.4 2952 96.1 74.3 2952G 93.5 39.9 2954 93.7 70.1 2954G 91.7 30.1 295575.8 30.0 2955G 71.8 ND 2964 92.1 31.4 2964G 94.6 43.0 2978 98.0 95.12978G 13.9 94.1 2982 92.8 78.5 2982G 41.9 52.4 2985 39.5 31.2 2985G 2.05.0 2987 81.7 67.8 2987G 26.6 29.3 2996 87.3 55.3 2996G 95.9 38.4 299793.4 70.6 2997G 9.7 7.5 3004 79.0 48.4 3004G 60.3 40.7 3005 97.4 93.53005G 77.5 75.6 3010 98.0 82.6 3010G 97.9 81.0 3011 87.4 42.8 3011G 83.541.7 3012 91.0 60.8 3012G 52.4 16.8 3013 80.3 65.8 3013G 17.5 15.4 301463.4 20.7 3014G 74.4 28.5 3015 89.1 55.7 3015G 58.8 17.3 3016 97.1 81.63016G 93.1 66.4 3017 94.8 70.2 3017G 87.9 40.8 3018 85.4 54.0 3018G 26.112.7 3019 99.3 92.4 3019G 99.3 88.1 3020 96.7 90.3 3020G 85.2 41.5 302174.5 26.1 3021G 81.1 27.4 3022 65.2 17.6 3022G 67.2 9.1 3023 71.4 28.53023G 73.8 29.7 3024 73.9 32.6 3024G 89.0 10.0 3025 70.7 15.6 3025G 76.724.3 3027 96.2 61.6 3027G 98.6 75.3 3028 92.4 29.0 3028G 87.3 28.8 30306.0 10.6 3030G 41.3 14.2 3032 76.5 31.4 3032G 17.7 11.0 3033 98.2 86.13033G 93.6 64.0 3036 74.7 32.7 3036G 90.1 51.2 3041 95.3 75.9 3041G 92.451.6 3042 88.1 73.3 3042G 60.9 25.2 3043 90.8 65.8 3043G 92.8 60.3

TABLE 8 Vκ3-20Jκ1 Common Light Chain Antibodies % Blocking of % Blockingof Antibody Antigen E-Labeled Beads Antigen E In Solution 2968 97.1 73.32968G 67.1 14.6 2969 51.7 20.3 2969G 37.2 16.5 2970 92.2 34.2 2970G 92.727.2 2971 23.4 11.6 2971G 18.8 18.9 2972 67.1 38.8 2972G 64.5 39.2 297377.7 27.0 2973G 51.1 20.7 2974 57.8 12.4 2974G 69.9 17.6 2975 49.4 18.22975G 32.0 19.5 2976 1.0 1.0 2976G 50.4 20.4

In the first LUMINEX™ experiment described above, 80 common light chainantibodies containing the Vκ1-39Jκ5 engineered light chain were testedfor their ability to block Ligand Y binding to Antigen E-labeled beads.Of these 80 common light chain antibodies, 68 demonstrated >50%blocking, while 12 demonstrated <50% blocking (6 at 25-50% blocking and6 at <25% blocking). For the 18 common light chain antibodies containingthe Vκ3-20Jκ1 engineered light chain, 12 demonstrated >50% blocking,while 6 demonstrated <50% blocking (3 at 25-50% blocking and 3 at <25%blocking) of Ligand Y binding to Antigen E-labeled beads.

In the second LUMINEX™ experiment described above, the same 80 commonlight chain antibodies containing the Vκ1-39Jκ5 engineered light chainwere tested for their ability to block binding of Antigen E to LigandY-labeled beads. Of these 80 common light chain antibodies, 36demonstrated >50% blocking, while 44 demonstrated <50% blocking (27 at25-50% blocking and 17 at <25% blocking). For the 18 common light chainantibodies containing the Vκ3-20Jκ1 engineered light chain, 1demonstrated >50% blocking, while 17 demonstrated <50% blocking (5 at25-50% blocking and 12 at <25% blocking) of Antigen E binding to LigandY-labeled beads.

The data of Tables 7 and 8 establish that the rearrangements describedin Tables 5 and 6 generated anti-Antigen E-specific common light chainantibodies that blocked binding of Ligand Y to its cognate receptorAntigen E with varying degrees of efficacy, which is consistent with theanti-Antigen E common light chain antibodies of Tables 5 and 6comprising antibodies with overlapping and non-overlapping epitopespecificity with respect to Antigen E.

Example 8 Determination of Blocking Ability of Antigen-Specific CommonLight Chain Antibodies by ELISA

Human common light chain antibodies raised against Antigen E were testedfor their ability to block Antigen E binding to a Ligand Y-coatedsurface in an ELISA assay.

Ligand Y was coated onto 96-well plates at a concentration of 2 μg/mLdiluted in PBS and incubated overnight followed by washing four times inPBS with 0.05% Tween-20. The plate was then blocked with PBS (IrvineScientific, Santa Ana, Calif.) containing 0.5% (w/v) BSA (Sigma-AldrichCorp., St. Louis, Mo.) for one hour at room temperature. In a separateplate, supernatants containing anti-Antigen E common light chainantibodies were diluted 1:10 in buffer. A mock supernatant with the samecomponents of the antibodies was used as a negative control. AntigenE-mmH (described above) was added to a final concentration of 0.150 nMand incubated for one hour at room temperature. The antibody/AntigenE-mmH mixture was then added to the plate containing Ligand Y andincubated for one hour at room temperature. Detection of Antigen E-mmHbound to Ligand Y was determined with Horse-Radish Peroxidase (HRP)conjugated to anti-Penta-His antibody (Qiagen, Valencia, Calif.) anddeveloped by standard colorimetric response using tetramethylbenzidine(TMB) substrate (BD Biosciences, San Jose, Calif.) neutralized bysulfuric acid. Absorbance was read at OD450 for 0.1 sec. Backgroundabsorbance of a sample without Antigen E was subtracted from allsamples. Percent blocking was calculated by division of thebackground-subtracted MFI of each sample by the adjusted negativecontrol value, multiplying by 100 and subtracting the resulting valuefrom 100.

Tables 9 and 10 show the percent blocking for all 98 anti-Antigen Ecommon light chain antibodies tested in the ELISA assay. ND: notdetermined under current experimental conditions.

TABLE 9 Vκ1-39Jκ5 Common Light Chain Antibodies % Blocking of AntibodyAntigen E In Solution 2948 21.8 2948G 22.9 2949 79.5 2949G 71.5 295080.4 2950G 30.9 2952 66.9 2952G 47.3 2954 55.9 2954G 44.7 2955 12.12955G 25.6 2964 34.8 2964G 47.7 2978 90.0 2978G 90.2 2982 59.0 2982G20.4 2985 10.5 2985G ND 2987 31.4 2987G ND 2996 29.3 2996G ND 2997 48.72997G ND 3004 16.7 3004G 3.5 3005 87.2 3005G 54.3 3010 74.5 3010G 84.63011 19.4 3011G ND 3012 45.0 3012G 12.6 3013 39.0 3013G 9.6 3014 5.23014G 17.1 3015 23.7 3015G 10.2 3016 78.1 3016G 37.4 3017 61.6 3017G25.2 3018 40.6 3018G 14.5 3019 94.6 3019G 92.3 3020 80.8 3020G ND 30217.6 3021G 20.7 3022 2.4 3022G 15.0 3023 9.1 3023G 19.2 3024 7.5 3024G15.2 3025 ND 3025G 13.9 3027 61.4 3027G 82.7 3028 40.3 3028G 12.3 3030ND 3030G 9.5 3032 ND 3032G 13.1 3033 77.1 3033G 32.9 3036 17.6 3036G24.6 3041 59.3 3041G 30.7 3042 39.9 3042G 16.1 3043 57.4 3043G 46.1

TABLE 10 Vκ3-20Jκ1 Common Light Chain Antibodies % Blocking of AntibodyAntigen E In Solution 2968 68.9 2968G 15.2 2969 10.1 2969G 23.6 297034.3 2970G 41.3 2971 6.3 2971G 27.1 2972 9.6 2972G 35.7 2973 20.7 2973G23.1 2974 ND 2974G 22.0 2975 8.7 2975G 19.2 2976 4.6 2976G 26.7

As described in this Example, of the 80 common light chain antibodiescontaining the Vκ1-39Jκ5 engineered light chain tested for their abilityto block Antigen E binding to a Ligand Y-coated surface, 22demonstrated >50% blocking, while 58 demonstrated <50% blocking (20 at25-50% blocking and 38 at <25% blocking). For the 18 common light chainantibodies containing the Vκ3-20Jκ1 engineered light chain, onedemonstrated >50% blocking, while 17 demonstrated <50% blocking (5 at25-50% blocking and 12 at <25% blocking) of Antigen E binding to aLigand Y-coated surface.

These results are also consistent with the Antigen E-specific commonlight chain antibody pool comprising antibodies with overlapping andnon-overlapping epitope specificity with respect to Antigen E.

Example 9 BIACORE™ Affinity Determination for Antigen-Specific CommonLight Chain Antibodies

Equilibrium dissociation constants (K_(D)) for selected antibodysupernatants were determined by SPR (Surface Plasmon Resonance) using aBIACORE™ T100 instrument (GE Healthcare). All data was obtained usingHBS-EP (10 mM Hepes, 150 mM NaCl, 0.3 mM EDTA, 0.05% Surfactant P20, pH7.4) as both the running and sample buffers, at 25° C. Antibodies werecaptured from crude supernatant samples on a CM5 sensor chip surfacepreviously derivatized with a high density of anti-human Fc antibodiesusing standard amine coupling chemistry. During the capture step,supernatants were injected across the anti-human Fc surface at a flowrate of 3 μL/min, for a total of 3 minutes. The capture step wasfollowed by an injection of either running buffer or analyte at aconcentration of 100 nM for 2 minutes at a flow rate of 35 μL/min.Dissociation of antigen from the captured antibody was monitored for 6minutes. The captured antibody was removed by a brief injection of 10 mMglycine, pH 1.5. All sensorgrams were double referenced by subtractingsensorgrams from buffer injections from the analyte sensorgrams, therebyremoving artifacts caused by dissociation of the antibody from thecapture surface. Binding data for each antibody was fit to a 1:1 bindingmodel with mass transport using BIAcore T100 Evaluation software v2.1.Results are shown in Tables 11 and 12.

TABLE 11 Vκ1-39Jκ5 Common Light Chain Antibodies 100 nM Antigen EAntibody K_(D) (nM) T_(1/2) (min) 2948 8.83 28 2948G 95.0 1 2949 3.57 182949G 6.37 9 2950 4.91 17 2950G 13.6 5 2952 6.25 7 2952G 7.16 4 29542.37 24 2954G 5.30 9 2955 14.4 6 2955G 12.0 4 2964 14.8 6 2964G 13.0 92978 1.91 49 2978G 1.80 58 2982 6.41 19 2982G 16.3 9 2985 64.4 9 2985G2.44 8 2987 21.0 11 2987G 37.6 4 2996 10.8 9 2996G 24.0 2 2997 7.75 192997G 151 1 3004 46.5 14 3004G 1.93 91 3005 2.35 108 3005G 6.96 27 30104.13 26 3010G 2.10 49 3011 59.1 5 3011G 41.7 5 3012 9.71 20 3012G 89.9 23013 20.2 20 3013G 13.2 4 3014 213 4 3014G 36.8 3 3015 29.1 11 3015G65.9 0 3016 4.99 17 3016G 18.9 4 3017 9.83 8 3017G 55.4 2 3018 11.3 363018G 32.5 3 3019 1.54 59 3019G 2.29 42 3020 5.41 39 3020G 41.9 6 302150.1 6 3021G 26.8 4 3022 25.7 17 3022G 20.8 12 3023 263 9 3023G 103 53024 58.8 7 3024G 7.09 10 3025 35.2 6 3025G 42.5 8 3027 7.15 6 3027G4.24 18 3028 6.89 37 3028G 7.23 22 3030 46.2 7 3030G 128 3 3032 53.2 93032G 13.0 1 3033 4.61 17 3033G 12.0 5 3036 284 12 3036G 18.2 10 30416.90 12 3041G 22.9 2 3042 9.46 34 3042G 85.5 3 3043 9.26 29 3043G 13.122

TABLE 12 Vκ3-20Jκ1 Common Light Chain Antibodies 100 nM Antigen EAntibody K_(D) (nM) T_(1/2) (min) 2968 5.50 8 2968G 305 0 2969 34.9 22969G 181 1 2970G 12.3 3 2971G 32.8 22 2972 6.02 13 2972G 74.6 26 29735.35 39 2973G 11.0 44 2974 256 0 2974G 138 0 2975 38.0 2 2975G 134 12976 6.73 10 2976G 656 8

The binding affinities of common light chain antibodies comprising therearrangements shown in Tables 5 and 6 vary, with nearly all exhibitinga K_(D) in the nanomolar range. The affinity data is consistent with thecommon light chain antibodies resulting from the combinatorialassociation of rearranged variable domains described in Tables 5 and 6being high-affinity, clonally selected, and somatically mutated. Coupledwith data previously shown, the common light chain antibodies describedin Tables 5 and 6 comprise a collection of diverse, high-affinityantibodies that exhibit specificity for one or more epitopes on AntigenE.

Example 10 Determination of Binding Specificities of Antigen-SpecificCommon Light Chain Antibodies by LUMINEX™ Assay

Selected anti-Antigen E common light chain antibodies were tested fortheir ability to bind to the ECD of Antigen E and Antigen E ECDvariants, including the cynomolgous monkey ortholog (Mf Antigen E),which differs from the human protein in approximately 10% of its aminoacid residues; a deletion mutant of Antigen E lacking the last 10 aminoacids from the C-terminal end of the ECD (Antigen E-ACT); and twomutants containing an alanine substitution at suspected locations ofinteraction with Ligand Y (Antigen E-Ala1 and AntigenE-Ala2). TheAntigen E proteins were produced in CHO cells and each contained amyc-myc-His C-terminal tag.

For the binding studies, Antigen E ECD protein or variant protein(described above) from 1 mL of culture medium was captured by incubationfor 2 hr at room temperature with 1×10⁶ microsphere (LUMINEX™) beadscovalently coated with an anti-myc monoclonal antibody (MAb 9E10,hybridoma cell line CRL-1729™; ATCC, Manassas, Va.). The beads were thenwashed with PBS before use. Supernatants containing anti-Antigen Ecommon light chain antibodies were diluted 1:4 in buffer and added to96-well filter plates. A mock supernatant with no antibody was used asnegative control. The beads containing the captured Antigen E proteinswere then added to the antibody samples (3000 beads per well) andincubated overnight at 4° C. The following day, the sample beads werewashed and the bound common light chain antibody was detected with aR-phycoerythrin-conjugated anti-human IgG antibody. The fluorescenceintensity of the beads (approximately 100 beads counted for eachantibody sample binding to each Antigen E protein) was measured with aLUMINEX™ flow cytometry-based analyzer, and the median fluorescenceintensity (MFI) for at least 100 counted beads per bead/antibodyinteraction was recorded. Results are shown in Tables 13 and 14.

TABLE 13 Vκ1-39Jκ5 Common Light Chain Antibodies Mean FluorescenceIntensity (MFI) Antigen Antigen Antigen Antigen Mf Antibody E-ECD E-ΔCTE-Ala1 E-Ala2 Antigen E 2948 1503 2746 4953 3579 1648 2948G 537 662 25812150 863 2949 3706 4345 8169 5678 5142 2949G 3403 3318 7918 5826 55142950 3296 4292 7756 5171 4749 2950G 2521 2408 7532 5079 3455 2952 33841619 1269 168 911 2952G 3358 1001 108 55 244 2954 2808 3815 7114 50393396 2954G 2643 2711 7620 5406 3499 2955 1310 2472 4738 3765 1637 2955G1324 1802 4910 3755 1623 2964 5108 1125 4185 346 44 2964G 4999 729 4646534 91 2978 6986 2800 14542 10674 8049 2978G 5464 3295 11652 8026 64522982 4955 2388 13200 9490 6772 2982G 3222 2013 8672 6509 4949 2985 1358832 4986 3892 1669 2985G 43 43 128 244 116 2987 3117 1674 7646 5944 25462987G 3068 1537 9202 6004 4744 2996 4666 1917 12875 9046 6459 2996G 27521736 8742 6150 4873 2997 5164 2159 12167 8361 5922 2997G 658 356 33922325 1020 3004 2794 1397 8542 6268 3083 3004G 2753 1508 8267 5808 43453005 5683 2221 12900 9864 5868 3005G 4344 2732 10669 7125 5880 3010 48291617 2642 3887 44 3010G 3685 1097 2540 3022 51 3011 2859 2015 7855 55133863 3011G 2005 1072 6194 4041 3181 3012 3233 2221 8543 5637 3307 3012G968 378 3115 2261 1198 3013 2343 1791 6715 4810 2528 3013G 327 144 13331225 370 3014 1225 1089 5436 3621 1718 3014G 1585 851 5178 3705 24113015 3202 2068 8262 5554 3796 3015G 1243 531 4246 2643 1611 3016 42202543 8920 5999 5666 3016G 2519 1277 6344 4288 4091 3017 3545 2553 87005547 5098 3017G 1972 1081 5763 3825 3038 3018 2339 1971 6140 4515 22933018G 254 118 978 1020 345 3019 5235 1882 7108 4249 54 3019G 4090 12704769 3474 214 3020 3883 3107 8591 6602 4420 3020G 2165 1209 6489 42952912 3021 1961 1472 6872 4641 2742 3021G 2091 1005 6430 3988 2935 30222418 793 7523 2679 36 3022G 2189 831 6182 3051 132 3023 1692 1411 57883898 2054 3023G 1770 825 5702 3677 2648 3024 1819 1467 6179 4557 24503024G 100 87 268 433 131 3025 1853 1233 6413 4337 2581 3025G 1782 7915773 3871 2717 3027 4131 1018 582 2510 22 3027G 3492 814 1933 2596 423028 4361 2545 9884 5639 975 3028G 2835 1398 7124 3885 597 3030 463 2771266 1130 391 3030G 943 302 3420 2570 1186 3032 2083 1496 6594 4402 24053032G 295 106 814 902 292 3033 4409 2774 8971 6331 5825 3033G 2499 12346745 4174 4210 3036 1755 1362 6137 4041 1987 3036G 2313 1073 6387 42433173 3041 3674 2655 8629 5837 4082 3041G 2519 1265 6468 4274 3320 30422653 2137 7277 5124 3325 3042G 1117 463 4205 2762 1519 3043 3036 21287607 5532 3366 3043G 2293 1319 6573 4403 3228

TABLE 14 Vκ3-20Jκ1 Common Light Chain Antibodies Mean FluorescenceIntensity (MFI) Antigen Antigen Antigen Antigen Mf Antibody E-ECD E-ΔCTE-Ala1 E-Ala2 Antigen E 2968 6559 3454 14662 3388 29 2968G 2149 375 9109129 22 2969 2014 1857 7509 5671 3021 2969G 1347 610 6133 4942 2513 29705518 1324 14214 607 32 2970G 4683 599 12321 506 31 2971 501 490 25062017 754 2971G 578 265 2457 2062 724 2972 2164 2158 8408 6409 3166 2972G1730 992 6364 4602 2146 2973 3527 1148 3967 44 84 2973G 1294 276 1603 2844 2974 1766 722 8821 241 19 2974G 2036 228 8172 135 26 2975 1990 14768669 6134 2468 2975G 890 315 4194 3987 1376 2976 147 140 996 1079 1812976G 1365 460 6024 3929 1625

The anti-Antigen E common light chain antibody supernatants exhibitedhigh specific binding to the beads linked to Antigen E-ECD. For thesebeads, the negative control mock supernatant resulted in negligiblesignal (<10 MFI) when combined with the Antigen E-ECD bead sample,whereas the supernatants containing anti-Antigen E common light chainantibodies exhibited strong binding signal (average MFI of 2627 for 98antibody supernatants; MFI >500 for 91/98 antibody samples).

As a measure of the ability of the selected anti-Antigen E common lightchain antibodies to identify different epitopes on the ECD of Antigen E,the relative binding of the antibodies to the variants were determined.All four Antigen E variants were captured to the anti-myc LUMINEX™ beadsas described above for the native Antigen E-ECD binding studies, and therelative binding ratios (MFI_(variant)/MFI_(Antigen E-ECD)) weredetermined. For 98 tested common light chain antibody supernatants shownin Tables 12 and 13, the average ratios(MFI_(variant)/MFI_(Antigen E-EDD)) differed for each variant, likelyreflecting different capture amounts of proteins on the beads (averageratios of 0.61, 2.9, 2.0, and 1.0 for Antigen E-ACT, Antigen E-Ala1,Antigen E-Ala2, and Mf Antigen E, respectively). For each proteinvariant, the binding for a subset of the 98 tested common light chainantibodies showed greatly reduced binding, indicating sensitivity to themutation that characterized a given variant. For example, 19 of thecommon light chain antibody samples bound to the Mf Antigen E withMFI_(variant)/MFI_(Antigen E-ECD) of <8%. Since many in this groupinclude high or moderately high affinity antibodies (5 with K_(D)<5 nM,15 with K_(D)<50 nM), it is likely that the lower signal for this groupresults from sensitivity to the sequence (epitope) differences betweennative Antigen E-ECD and a given variant rather than from loweraffinities.

These data establish that the common light chain antibodies described inTables 5 and 6 represent a diverse group of Antigen-E-specific commonlight chain antibodies that specifically recognize more than one epitopeon Antigen E.

Example 11 Light Chain Shuffling in Common Light Chain Antibodies

Heavy chains of selected antigen-specific common light chain antibodieswere tested for binding to Antigen E after repairing the heavy chainswith either a germline Vκ1-39Jκ5 or a germline Vκ3-20Jκ1 engineeredlight chain (as described in Example 1).

Briefly, 247 heavy chains of Antigen E-specific common light chainantibodies (Vκ1-39Jκ5 and Vκ3-20Jκ1) were transfected with either agermline Vκ1-39 or a germline Vκ3-20 engineered light chain andrescreened for binding to Antigen E by a LUMINEX™ assay (as described inExample 7 and Example 10). Binding to Antigen E was confirmed byBIACORE™ (as described in Example 9). The results are shown in Table 15.

As shown in this Example, twenty-eight common light chain antibodiesspecific for Antigen E were capable of binding to Antigen E whenrepaired with a germline form of the light chain.

TABLE 15 Original Repaired No. No. Confirmed Light Chain Light ChainTested Binders 1-39 1-39 198 23 3-20 3-20 49 5

Example 12 Heavy Chain Gene Usage and Somatic Hypermutation Frequency inCommon Light Chain Antibodies

Heavy and light chain sequences (>6000) of antibodies raised inVELCOIMMUNE® mice (e.g., U.S. Pat. No. 6,596,541 and U.S. Pat. No.7,105,348) were compiled with heavy and light chain sequences (>600) ofcommon light chain antibodies obtained by a multi-antigen immunizationscheme employing the engineered light chain mice (described above) tocompare heavy chain gene segment usage and somatic hypermutationfrequencies of the antibody chains.

Heavy Chain Gene Usage.

Heavy and light chain sequences obtained from VELOCIMMUNE® micecontaining a replacement of the endogenous mouse heavy chain locus withhuman V_(H), D_(H), and J_(H) gene segments and a replacement of theendogenous mouse κ light chain locus with either the engineered germlineVκ1-39Jκ5 human light chain region or the engineered germline Vκ3-20Jκ1human light chain region (as described in Example 2) immunized with ahuman cell-surface receptor (Antigen E), a heterodimer of two humancell-surface glycoproteins (Antigen F), a human cytokine receptor(Antigen G) and a human tumor differentiation antigen (Antigen H) wereanalyzed for heavy chain gene segment usage and V_(H) and J_(H) genesegments were recorded. Results are shown in Tables 16-18. Percentagesin Tables 16-18 represent rounded values and in some cases may not equal100% when added together.

Table 16 sets forth the percent heavy chain family usage for antibodiesfrom VELCOIMMUNE® mice (VI), antibodies from VELCOIMMUNE® mice having acognate Vκ1-39 light chain (VI-Vκ1-39), antibodies from Vκ1-39engineered light chain mice (Vκ1-39), antibodies from VELCOIMMUNE® micehaving a cognate Vκ3-20 light chain (VI-Vκ3-20), and antibodies fromVκ3-20 engineered light chain mice (Vκ3-20). Table 17 sets forth thepercent V_(H) and J_(H) gene usage for antibodies from VELCOIMMUNE® mice(VI), antibodies from VELCOIMMUNE® mice having a cognate Vκ1-39 lightchain (VI-Vκ1-39), antibodies from Vκ1-39 engineered light chain mice(Vκ1-39), antibodies from VELCOIMMUNE® mice having a cognate Vκ3-20light chain (VI-Vκ3-20), and antibodies from Vκ3-20 engineered lightchain mice (Vκ3-20). Table 18 sets forth the percent V_(H) gene usagefor antibodies from Vκ1-39 engineered light chain mice (Vκ1-39 Mice)from each immunization group (Antigens E, F, G and H) and the percentV_(H) gene usage for antibodies from Vκ3-20 engineered light chain mice(Vκ3-20 Mice) from selected immunization groups (Antigens E and G).

As shown in this Example, heavy chain gene usage for antigens tested inVκ1-39Jκ5-engineered light chain mice was characterized by apreponderance of V_(H) family III subgroups (V_(H)3-7, V_(H)3-9,V_(H)3-11, V_(H)3-13, V_(H)3-20, V_(H)3-23, V_(H)3-30, V_(H)3-33 andV_(H)3-48). Notable usage of other V_(H) family subgroups wascharacterized by usage of V_(H)1-18, V_(H)1-69, V_(H)2-5, V_(H)4-59 andV_(H)6-1. For antigens tested in Vκ3-20Jκ1 engineered light chain mice,heavy chain gene usage was characterized by a preponderance of V_(H)family III, V_(H) family IV and V_(H) family V subgroups (V_(H)3-11,V_(H)3-30, V_(H)3-33, V_(H)4-39, V_(H)4-59 and V_(H)5-51). Notable usageof other V_(H) family subgroups was characterized by usage of V_(H)1-18,V_(H)1-69, V_(H)2-70 and V_(H)6-1.

Somatic Hypermutation Frequency.

Heavy and light chains from antibodies raised in VELCOIMMUNE® mice andthe engineered light chain mice (described above) were aligned togermline sequences according to the heavy and light chain gene usagedemonstrated for each heavy and/or light chain. Amino acid changes foreach framework region (FW) and complementarity determining region (CDR)for both heavy and light chain of each sequence were calculated. Resultsare shown in Tables 19-22. Percentages in Tables 21-24 represent roundedvalues and in some cases may not equal 100% when added together.

Table 19 sets forth the number of amino acid (AA) changes observed ineach FW and CDR region of heavy chains of antibodies from VELCOIMMUNE®mice, heavy chains of antibodies from Vκ1-39 engineered light chain mice(Vκ1-39 Mice) and heavy chains of antibodies from Vκ3-20 engineeredlight chain mice (Vκ3-20 Mice). Table 20 sets forth the number of aminoacid (AA) changes observed in each FW and CDR region of light chains ofantibodies from VELCOIMMUNE® mice, the light chain of antibodies fromVκ1-39 engineered mice (Vκ1-39 Mice) and the light chain of antibodiesfrom Vκ3-20 engineered mice (Vκ3-20 Mice). Table 21 sets forth thenumber of amino acid (AA) changes observed in each FW and CDR region ofheavy chains of antibodies from Vκ1-39 engineered light chain mice(Vκ1-39 Mice) for selected immunization groups (Antigens E, F and H).Table 22 sets forth the number of amino acid (AA) changes observed ineach FW and CDR region of heavy chains of antibodies from Vκ3-20engineered light chain mice (Vκ3-20 Mice) for selected immunizationgroups (Antigens E and G).

TABLE 16 V_(H) VI - VI - Family VI Vκ1-39 Vκ1-39 Vκ3-20 Vκ3-20 1 9.014.8 3.3 7.1 4.9 2 2.2 1.8 4.6 0 1.6 3 77.8 69.8 77.3 61.4 29.5 4 8.48.3 11.2 27.1 39.3 5 0.9 0 0.7 4.3 23.0 6 1.7 5.3 3.0 0 1.6

TABLE 17 VI - VI - VI Vκ1-39 Vκ1-39 Vκ3-20 Vκ3-20 V_(H) Gene 1-2  3.98.3 0 2.9 0 1-3  0 0 0 0 0 1-8  1.3 0.6 0 1.4 0 1-18 3.0 0.6 1.3 2.1 1.61-24 0.4 3.6 0 0.7 0 1-46 0.1 0 0 0 0 1-58 0 0 0 0 0 1-69 0.3 1.8 2.0 03.3 2-5  1.9 0 4.6 0 0 2-26 0.2 1.8 0.0 0 0 2-70 0.1 0 0 0 1.6 3-7  3.014.8 0 1.4 0 3-9  8.5 3.6 29.6 16.4 0 3-11 5.4 10.7 0 7.1 1.6 3-13 3.21.8 0.7 2.1 0 3-15 4.0 4.7 0.3 0.7 0 3-20 1.0 0.6 0.3 5.0 0 3-21 0.8 0.60 2.1 0 3-23 20.4 8.9 3.3 8.6 0 3-30 17.6 4.1 35.2 12.9 1.6 3-33 12.614.8 0 5.0 26.2 3-43 0.2 0.6 0 0 0 3-48 0.8 1.2 7.2 0 0 3-53 0.3 3.6 0.30 0 3-64 0 0 0.3 0 0 3-72 0 0 0 0 0 3-73 0 0 0 0 0 4-31 2.7 0 0.7 8.6 04-34 1.8 0.6 0.3 14.3 0 4-39 1.6 0.6 3.0 2.1 14.8 4-59 2.3 7.1 7.2 2.124.6 5-51 0.9 0 0.7 4.3 23.0 6-1  1.7 5.3 3.0 0 1.6 J_(H) Gene 1 1.5 1.27.1 0 0 2 4.5 2.4 0.7 5.0 26.9 3 10.5 16.6 13.1 13.6 26.9 4 44.0 34.332.3 50.7 9.6 5 9.6 10.1 16.8 7.9 1.9 6 29.7 35.5 30.0 22.9 34.6

TABLE 18 Vκ1-39 Mice Vκ3-20 Mice Antigen Antigen Antigen Antigen V_(H)Gene E F G Antigen H E Antigen G 1-2 0 0 0 0 0 0 1-3 0 0 0 0 0 0 1-8 0 00 0 0 0 1-18 0 0 0 8.3 0 3.1 1-24 0 0 0 0 0 0 1-46 0 0 0 0 0 0 1-58 0 00 0 0 0 1-69 2.9 0 25.0 0 0 6.3 2-5 8.2 0 0 0 0 0 2-26 0 0 0 0 0 0 2-700 0 0 0 0 3.1 3-7 0 0 0 0 0 0 3-9 1.2 98.8 0 14.6 0 0 3-11 0 0 0 0 0 3.13-13 0.6 0 25.0 0 0 0 3-15 0 1.2 0 0 0 0 3-20 0 0 25.0 0 0 0 3-21 0 0 00 0 0 3-23 4.1 0 25.0 4.2 0 0 3-30 62.9 0 0 0 3.4 0 3-33 0 0 0 0 13.837.5 3-43 0 0 0 0 0 0 3-48 0.6 0 0 43.8 0 0 3-53 1.6 0 0 0 0 0 3-64 1.60 0 0 0 0 3-72 0 0 0 0 0 0 3-73 0 0 0 0 0 0 4-31 0 0 0 4.2 0 0 4-34 0 00 2.1 0 0 4-39 5.3 0 0 0 31.0 0 4-59 11.8 0 0 4.2 3.4 43.8 5-51 1.2 0 00 48.3 0 6-1 0 0 0 18.8 0 3.1

TABLE 19 # AA Changes FW1 CDR1 FW2 CDR2 FW3 CDR3 Heavy Chains ofAntibodies from VELCOIMMUNE ® Mice 0 63 32 36 26 12 82 1 23 32 41 31 2217 2 9 25 17 23 27 1 3 4 10 5 16 13 0 4 0 1 1 3 12 0 >5 1 0 0 1 14 0Heavy Chains of Antibodies from Vκ1-39 Mice 0 65 8 34 30 9 37 1 25 26 3534 19 54 2 9 44 23 20 33 9 3 1 19 8 12 22 0 4 0 3 0 5 11 0 >5 1 0 0 0 70 Heavy Chains of Antibodies from Vκ3-20 Mice 0 57 8 54 16 8 93 1 41 4334 39 21 7 2 2 25 10 18 20 0 3 0 15 2 21 13 0 4 0 10 0 3 20 0 >5 0 0 0 218 0

TABLE 20 # AA Changes FW1 CDR1 FW2 CDR2 FW3 CDR3 Light Chains ofAntibodies from VELCOIMMUNE ® Mice 0 65 24 49 60 33 23 1 24 20 34 31 2738 2 9 27 16 9 18 28 3 1 20 1 0 14 7 4 0 7 0 0 4 3 >5 1 1 0 0 3 0 LightChains of Antibodies from Vκ1-39 Mice 0 91 75 80 90 71 63 1 9 19 17 1021 27 2 0 5 1 1 5 8 3 0 0 1 0 2 1 4 0 0 0 0 2 1 >5 0 0 0 0 0 0 LightChains of Antibodies from Vκ3-20 Mice 0 90 47 93 97 63 57 1 10 27 3 3 2043 2 0 27 3 0 17 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 >5 0 0 0 0 0 0

TABLE 21 # AA Changes FW1 CDR1 FW2 CDR2 FW3 CDR3 Heavy Chains ofAnti-Antigen E Antibodies from Vκ1-39 Mice 0 75 8 49 41 14 36 1 21 25 3335 25 52 2 4 43 14 18 28 12 3 0 20 4 5 16 0 4 0 5 0 1 12 0 >5 1 0 0 0 50 Heavy Chains of Anti-Antigen F Antibodies from Vκ1-39 Mice 0 52 0 6 62 15 1 35 24 32 35 15 78 2 11 59 46 22 49 7 3 0 17 16 24 29 0 4 0 0 0 124 0 >5 1 0 0 0 1 0 Heavy Chains of Anti-Antigen H Antibodies from Vκ1-39Mice 0 54 21 29 33 4 77 1 17 35 50 27 6 23 2 23 21 15 21 25 0 3 6 21 415 27 0 4 0 2 2 2 15 0 >5 0 0 0 2 23 0

TABLE 22 # AA Changes FW1 CDR1 FW2 CDR2 FW3 CDR3 Heavy Chains ofAnti-Antigen E Antibodies from Vκ3-20 Mice 0 79 17 62 24 17 90 1 21 2834 55 31 10 2 0 28 3 21 24 0 3 0 14 0 0 10 0 4 0 14 0 0 3 0 >5 0 0 0 014 0 Heavy Chains of Anti-Antigen G Antibodies from Vκ3-20 Mice 0 38 047 9 0 97 1 59 56 34 25 13 3 2 3 22 16 16 16 0 3 0 16 3 41 16 0 4 0 6 06 34 0 >5 0 0 0 3 22 0

Example 13 Binding Affinity of Bispecific Antibodies Having UniversalLight Chains

Fully human bispecific antibodies were constructed from cloned humanheavy chain variable regions of selected monospecific anti-Antigen Ecommon light chain antibodies (described in Example 5) using standardrecombinant DNA techniques known in the art. Table 23 sets forth thepairing of human heavy chains (HC-1 and HC-2) from selected parentalmonospecific antibodies; each pair employed with a germline rearrangedhuman Vκ1-39/Jκ1 light chain for construction of each bispecificantibody.

Binding of bispecific or parental monospecific anti-Antigen E antibodiesto the extracellular domain (ECD) of Antigen E was determined using areal-time surface plasmon resonance biosensor assay on a BIACORE™ 2000instrument (GE Healthcare). A CM5 BIACORE™ sensor surface derivatizedwith anti-c-myc-specific monoclonal antibody (Clone#9E10) using EDC-NHSchemistry was used to capture the C-terminal myc-myc-hexahistidinetagged ECD of Antigen E (AntigenE-mmh). Around 190 RUs of AntigenE-mmhwas captured on the BIACORE™ sensor surface, followed by the injectionof 300 nM and 50 nM concentrations of different bispecific or parentalmonospecific anti-Antigen E antibodies at a flow rate of 50 μl/min. Theexperiment was performed at 25° C. in HBST running buffer (0.01 M HEPESpH 7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20). The amount ofantibody binding to AntigenE-mmh surface at 300 nM concentration wasrecorded three seconds before the end of antibody injection and plotted.

Table 24 and FIG. 8 set forth the binding responses (BIACORE™ units; RU)observed for each bispecific antibody (BsAb) and monospecific parentalantibody (PAb-1, PAb-2). Since each antibody was injected undersaturating conditions over an identical AntigenE-mmh surface, thebinding response reflects the binding stoichiometry for each antibodybinding to the antigen capture surface.

As shown in this Example, the observed binding response for eachbispecific antibody was approximately 2-fold greater than the bindingresponse for each parental monospecific antibody (Table 24 and FIG. 8),demonstrating functional construction of bispecific antibodies usingheavy chains of antigen-specific monoclonal antibodies and a commonlight chain where each Fab arm in the bispecific antibody molecule bindssimultaneously to distinct epitopes on the extracellular domain of acell surface receptor (Antigen E; see FIG. 7B, bottom left).

TABLE 23 Bispecific Antibody Parent HC-1 Parent HC-2 3108 2952 2978 31092978 3022 3111 2952 3005 3112 3022 3005

TABLE 24 Binding Response (RU) Bispecific Antibody PAb-1 PAb-2 BsAb 3108236 229 485 3109 236 197 408 3111 202 229 435 3112 202 197 345

Example 14 Generation and Analysis of Mice Expressing Two Human LightChains

Using the methods described above in Example 2, two additionalengineered light chain loci containing two human Vκ gene segments (e.g.,a human Vκ1-39 and human Vκ3-20 gene segment) were constructed (FIG. 9).One engineered light chain locus contained two human Vκ gene segmentsand five human Jκ gene segments in unrearranged configuration (DLC-5J).The second engineered light chain locus contained two human Vκ genesegments and one human Jκ gene segment in unrearranged configuration(DLC-1J). For each of the two additional engineered light chain loci,the human gene segments were flanked 3′ with recombination signalsequences to allow for in vivo rearrangement of the human gene segmentsin B cells.

Modified BAC DNA clones separately containing each of the engineeredlight chain loci operably linked to mouse sequences (i.e., upstream anddownstream sequences of the endogenous immunoglobulin κ light chainlocus) were confirmed by PCR using primers located at sequences withineach engineered light chain locus containing the two human Vκ genesegments followed by electroporation into ES cells to create mice thatexpress either of the two human Vκ gene segments (as described above).Positive ES cell clones that contain either of the engineered lightchain loci described above were confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered light chain loci(as described above). Confirmed ES cell clones were then used to implantfemale mice to give rise to a litter of pups expressing a human lightchain variable domain fused with a mouse Cκ domain, referred to hereinas Dual Light Chain (DLC) mice.

Alternatively, ES cells bearing the engineered light chain locus may betransfected with a construct that expresses FLP in order to remove theFRTed neomycin cassette introduced by the targeting construct.Optionally, the neomycin cassette is removed by breeding to mice thatexpress FLP recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, theneomycin cassette is retained in the mice.

Flow Cytometry.

B cell populations and B cell development in DLC mice were validated byflow cytometry analysis of splenocyte and bone marrow preparations. Cellsuspensions from mice homozygous for two human Vκ gene segments and fivehuman Jκ gene segments (n=4), mice homozygous for two human Vκ genesegments and one human Jκ gene segment (n=4), and wild type mice (n=4)were made using standard methods (described above) and stained withfluorescently labeled antibodies (as described in Example 3).

Briefly, 1×10⁶ cells were incubated with anti-mouse CD16/CD32 (clone2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling withthe following antibody cocktail for 30 minutes on ice: APC-H7 conjugatedanti-mouse CD19 (clone 1D3, BD Pharmigen), Pacific Blue conjugatedanti-mouse CD3 (clone 17A2, BioLegend), FITC conjugated anti-mouse Igκ(clone 187.1, BD Pharmigen) or anti-mouse CD43 (clone 1B11, BioLegend),PE conjugated anti-mouse Igλ (clone RML-42, BioLegend) or anti-mousec-kit (clone 2B8, BioLegend), PerCP-Cy5.5 conjugated anti-mouse IgD(BioLegend), PE-Cy7 conjugated anti-mouse IgM (clone 11/41,eBioscience), APC conjugated anti-mouse B220 (clone RA3-6B2,eBioscience). Following staining, cells were washed and fixed in 2%formaldehyde. Data acquisition was performed on an LSRII flow cytometerand analyzed with FlowJo (Tree Star, Inc.). Gating: total B cells(CD19⁺CD3⁻), Igκ⁺ B cells (Igκ⁺Igλ⁻CD19⁺CD3⁻), Igλ⁺ B cells(Igκ⁻Igλ⁺CD19⁺CD3⁻). Results for the bone marrow compartment are shownin FIG. 10A-FIG. 12B. Results for the splenic compartment are shown inFIG. 13A-FIG. 16.

As shown in this Example, DLC-5J mice demonstrate normal B cellpopulations within the splenic and bone marrow compartments (FIG.10A-16). DLC-5J mice demonstrated immature, mature and pre/pro B cellpopulations within the bone marrow compartment that are substantiallythe same as observed in wild-type litter mates. In fact, the DLC-5Jlocus was capable of competing with the endogenous λ light chain locusto yield a κ:λ ratio that is substantially the same as that observed inwild-type mice (FIG. 14B). Also, DLC-5J mice demonstrate a normalperipheral B cell development as progression of B cells through variousstages in the splenic compartment (e.g., immature, mature, T1, T2 T3,marginal zone precursor, marginal zone, follicular-I, follicular-II,etc.) occurs in a manner substantially the same as observed in wild typemice (FIG. 15A-16). In contrast, DLC-1J mice demonstrated a loweroverall number of B cells and an increased λ light chain usage ascompared to the engineered κ light chain (data not shown).

Dual Light Chain Expression.

Expression of both human Vκ gene segments was analyzed in homozygousmice using a quantitative PCR assay in accordance with in Example 3.Briefly, CD19⁺ B cells were purified from bone marrow and whole spleensof wild type mice, mice homozygous for a replacement of the mouse heavychain and κ light chain variable loci with corresponding human heavychain and κ light chain variable region loci (Hκ), as well as micehomozygous for an engineered κ light chain loci containing two human Vκgene segments and either five human Jκ gene segments (DLC-5J) or onehuman Jκ gene segment (DLC-1J). Relative expression was normalized toexpression of mouse Cκ region (n=3 to 5 mice per group). Results areshown in FIG. 17 and FIG. 18.

Expression of light chains containing a rearranged human Vκ3-20 or humanVκ1-39 gene segment were detected in both the bone marrow and spleen ofDLC-5J and DLC-1J mice (FIG. 17 and FIG. 18). In the bone marrowcompartment, expression of both human Vκ3-20-derived and humanVκ1-39-derived light chains in both strains of DLC mice wassignificantly higher as compared to mice comprising a replacement ofmouse Vκ and Jκ gene segment with corresponding human Vκ and Jκ genesegments (Hκ; FIG. 17). Human Vκ3-20-derived light chain expression wasobserved at about six-fold (DLC-5J) to fifteen-fold (DLC-1J) higher thanin Hκ mice. DLC-1J mice demonstrated about two-fold higher expression ofhuman Vκ3-20-derived light chains over DLC-5J mice in the bone marrowcompartment. Human Vκ1-39-derived light chain expression was observed atabout six-fold (DLC-5J) to thirteen-fold (DLC-1J) higher than in Hκmice. DLC-1J mice demonstrated about two-fold higher expression of humanVκ1-39-derived light chains over DLC-5J mice in the bone marrowcompartment.

In the splenic compartment, expression of both human Vκ3-20-derived andhuman Vκ1-39-derived light chains in both strains of DLC mice wassignificantly higher as compared to Hκ mice (FIG. 18). HumanVκ3-20-derived light chain expression was observed at about four-fold(DLC-5J) and eight-fold (DLC-1J) higher than in Hκ mice. DLC-1J micedemonstrated about two-fold higher expression of human Vκ3-20-derivedlight chains over DLC-5J mice in the splenic compartment. HumanVκ1-39-derived light chain expression was observed at about four-fold(DLC-5J) to five-fold (DLC-1J) higher than in Hκ mice. DLC-1J micedemonstrated similar expression of human Vκ1-39-derived light chains ascompared to DLC-5J mice in the splenic compartment.

Human V_(L)/J_(L) Usage in DLC-5J.

Mice. Mice homozygous for two unrearranged human Vκ gene segments andfive unrearranged human Jκ gene segments (DLC-5J) were analyzed forhuman Vκ/Jκ gene segment usage in splenic B cells byreverse-transcriptase polymerase chain reaction (RT-PCR).

Briefly, spleens from homozygous DLC-5J (n=3) and wild type (n=2) micewere harvested and meshed in 10 mL of RPMI 1640 (Sigma) containing 10%heat-inactivated fetal bovine serum using frosted glass slides to createsingle cell suspensions. Splenocytes were pelleted with a centrifuge(1200 rpm for five minutes) and red blood cells were lysed in 5 mL ofACK lysing buffer (GIBCO) for three minutes. Splenocytes were dilutedwith PBS (Irvine Scientific), filtered with a 0.7 μm cell strainer andcentrifuged again to pellet cells, which was followed by resuspension in1 mL of PBS.

RNA was isolated from pelleted splenocytes using AllPrep DNA/RNA minikit (Qiagen) according to manufacturer's specifications. RT-PCR wasperformed on splenocyte RNA using 5′ RACE (Rapid Amplification of cDNAends) System with primers specific for the mouse Cκ gene according tomanufacturer's specifications (Invitrogen). The primers specific for themouse Cκ gene were 3′ mlgκC RACE1 (AAGAAGCACA CGACTGAGGC AC; SEQ ID NO:34) and mlgκC3′-1 (CTCACTGGAT GGTGGGAAGA TGGA; SEQ ID NO: 35). PCRproducts were gel-purified and cloned into pCR®2.1-TOPO® vector (TOPO®TA Cloning® Kit, Invitrogen) and sequenced with M13 Forward (GTAAAACGACGGCCAG; SEQ ID NO: 36) and M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:37) primers located within the vector at locations flanking the cloningsite. Ten clones from each spleen sample were sequenced. Sequences werecompared to the mouse and human immunoglobulin sets from theIMGT/V-QUEST reference directory sets to determine Vκ/Jκ usage. Table 25sets forth the Vκ/Jκ combinations for selected clones observed in RT-PCRclones from each splenocyte sample. Table 26 sets forth the amino acidsequence of the human Vκ/human Jκ and human Jκ/mouse Cκ junctions ofselected RT-PCR clones from DLC-5J homozygous mice. Lower case lettersindicate mutations in the amino acid sequence of the variable region ornon-template additions resulting from N and/or P additions duringrecombination.

As shown in this Example, mice homozygous for two unrearranged human Vκgene segments and five unrearranged human Jκ gene segments (DLC-5J)operably linked to the mouse Cκ gene are able to productively recombineboth human Vκ gene segments to multiple human Jκ gene segments toproduce a limited immunoglobulin light chain repertoire. Among therearrangements in DLC-5J homozygous mice shown in Table 25, unique humanVκ/Jκ rearrangements were observed for Vκ1-39/Jκ2 (1), Vκ1-39/Jκ3 (1),Vκ3-20/Jκ1 (7), Vκ3-20/Jκ2 (4) and Vκ3-20/Jκ3 (1). Further, such uniquerearrangements demonstrated junctional diversity through the presence ofunique amino acids within the CDR3 region of the light chain (Table 26)resulting from either mutation and/or the recombination of the human Vκand Jκ gene segments during development. All the rearrangements showedfunctional read through into mouse Cκ(Table 26).

Taken together, these data demonstrate that mice engineered to present achoice of no more than two human V_(L) gene segments, both of which arecapable of rearranging (e.g., with one or more and, in some embodiments,up to five human J_(L) gene segments) and encoding a human V_(L) domainof an immunoglobulin light chain have B cell numbers and developmentthat is nearly wild-type in all aspects. Such mice produce a collectionof antibodies having immunoglobulin light chains that have one of twopossible human V_(L) gene segments present in the collection. Thiscollection of antibodies is produced by the mouse in response to antigenchallenge and are associated with a diversity of reverse chimeric (humanvariable/mouse constant) heavy chains.

TABLE 25 Mouse ID No. Genotype Clone Vκ/Jκ Combination 1089451 DLC-5J1-2 1-39/3 1-4 3-20/2 1-7 3-20/1 1-8 3-20/2 1089452 DLC-5J 2-2 3-20/12-3 3-20/1 2-6 3-20/2 2-8 3-20/2 2-9 3-20/1  2-10 1-39/2 1092594 DLC-5J3-1 3-20/1 3-2 3-20/1 3-4 3-20/1 3-6 3-20/3 3-9 3-20/2 1092587 WT 1-119-93/1  1-2 6-25/1 1-3 4-91/5 1-5 3-10/4 1-6 4-86/4 1-8 19-93/1   1-1019-93/2  1092591 WT 2-1 19-93/1  2-3 6-20/5 2-4 6-25/5 2-5  1-117/1 2-68-30/1 2-7 8-19/2 2-8 8-30/1  2-10  1-117/1

TABLE 26 Sequence of hVκ/hJκ/mCκ Junction Clone Vκ/Jκ(CDR3 underlined, mlgκC italics) SEQ ID NO: 2-10 1-39/2QPEDFATYYCQQSYSTPYTFGQGTKLEIKRADAAPTVSI 38 1-2 1-39/3QPEDFATYYCQQSYSTPFTFGPGTKVDIKRADAAPTVSI 39 1-7 3-20/1EPEDFAVYYCQQYGSSPrTFGQGTKVEIKRADAAPTVSI 40 2-2 3-20/1EPEDFAVYYCQQYGSSrTFGQGTKVEIKRADAAPTVSI 41 2-3 3-20/1EPEDFAVYYCQQYGSSPWTFGQGTKVEIKRADAAPTVSI 42 2-9 3-20/1dPEDFAVYYCQQYGSSPrTFGQGTKVEIKRADAAPTVSI 44 3-1 3-20/1EPEDFAVYYCQQYGSSPrTFGQGTKVEIKRADAAPTVSI 45 3-2 3-20/1EPEDFAVYYCQQYGSSPWTFGQGTKVEIKRADAAPTVSI 46 3-4 3-20/1EPEDFAVYYCQQYGSSPPTFGQGTKVEIKRADAAPTVSI 47 3-9 3-20/2EPEDFAVYYCQQYGSSPYTFGQGTKLEIKRADAAPTVSI 48 3-6 3-20/3EPEDFAVYYCQQYGSSiFTFGPGTKVDIKRADAAPTVSI 49

What is claimed is:
 1. A mouse comprising no more than two human V_(L)gene segments and two or more human J_(L) gene segments operably linkedto a mouse or rat light chain constant region; and one or more humanV_(H), one or more human D_(H), and one or more human J_(H) genesegments operably linked to a non-human constant region; wherein thehuman gene segments are capable rearranging and encoding human variabledomains of an antibody, and further wherein the mouse does not comprisean endogenous V_(L) gene segment that is capable of rearranging to forman immunoglobulin light chain.
 2. The mouse of claim 1, wherein thelight chain constant region is a rat Cκ region.
 3. The mouse of claim 1,wherein the light chain constant region is a mouse Cκ region.
 4. Themouse of claim 1, wherein the mouse comprises five human J_(L) genesegments.
 5. The mouse of claim 4, wherein the five human J_(L) genesegments are human Jκ1, Jκ1 Jκ3, Jκ4 and Jκ5.
 6. The mouse of claim 1,wherein the no more than two human V_(L) gene segments are selected froma human Vκ1-39 gene segment, a human Vκ3-20 gene segment, and acombination thereof.
 7. The mouse of claim 6, wherein the no more thantwo human V_(L) gene segments are a human Vκ1-39 gene segment and ahuman Vκ3-20 gene segment.
 8. The mouse of claim 1, wherein the no morethan two human V_(L) gene segments and two or more human J_(L) genesegments are present at an endogenous light chain locus.
 9. The mouse ofclaim 1, wherein the one or more human V_(H), one or more human D_(H),and one or more human J_(H) gene segments are operably linked to a mouseconstant region.
 10. The mouse of claim 1, wherein the mouse comprises afunctional λ light chain locus.
 11. The mouse of claim 1, wherein themouse comprises a nonfunctional λ light chain locus.
 12. The mouse ofclaim 1, wherein the mouse comprises pro B cell population in the bonemarrow within the range of about 2.5×10⁴ to about 1.5×10⁵ cellscharacterized by CD19⁺, CD43⁺, c-kit⁺ expression.
 13. The mouse of claim1, wherein the mouse comprises a pre B cell population in the bonemarrow within in the range of about 1×10⁶ to about 2×10⁶ cellscharacterized by CD19⁺, CD43⁻, c-kit⁻ expression.
 14. The mouse of claim1, wherein the mouse comprises an immature B cell population in the bonemarrow within the range of about 5×10⁵ to about 7×10⁵ cellscharacterized by IgM⁺, B220^(int) expression.
 15. The mouse of claim 1,wherein the mouse comprises a mature B cell population in the bonemarrow within the range of about 3×10⁴ to about 1.5×10⁶ cellscharacterized by IgM⁺, B220^(hi) expression.
 16. The mouse of claim 1,wherein the mouse comprises a total CD19⁺ B cell population in the bonemarrow within the range of about 1×10⁶ to about 3×10⁶ cells.
 17. Themouse of claim 1, wherein the mouse comprises a CD19⁺ splenic B cellpopulation within the range of about 2×10⁶ to about 7×10⁶ cells.
 18. Themouse of claim 1, wherein the mouse comprises a CD19⁺, IgD^(hi),IgM^(lo) splenic B cell population within the range of about 1×10⁶ toabout 4×10⁶ cells.
 19. The mouse of claim 1, wherein the mouse comprisesa CD19⁺, IgD^(lo), IgM^(hi) splenic B cell population within the rangeof about 9×10⁵ to about 2×10⁶ cells.
 20. The mouse of claim 1, whereinthe mouse comprises a transitional T1 splenic B cell population withinthe range of about 2×10⁶ to about 7×10⁶ cells characterized by CD93⁺B220⁺ IgM^(hi) CD23⁻ expression.
 21. The mouse of claim 1, wherein themouse comprises a transitional T2 splenic B cell population within therange of about 1×10⁶ to about 7×10⁶ cells characterized by CD93⁺ B220⁺IgM^(hi)CD23⁺ expression.
 22. The mouse of claim 1, wherein the mousecomprises a transitional T3 splenic B cell population within the rangeof about 1×10⁶ to about 4×10⁶ cells characterized by CD93⁺ B220⁺IgM^(hi) CD23⁺ expression.
 23. The mouse of claim 1, wherein the mousecomprises a marginal zone splenic B cell population within the range ofabout 1×10⁶ to about 3×10⁶ cells characterized by CD93⁻ B220⁺IgM^(hi)CD21/35^(hi) CD23⁻ expression.
 24. The mouse of claim 1, whereinthe mouse comprises a follicular type 1 (FO-I) splenic B cell populationwithin the range of about 3×10⁶ to about 1.5×10⁷ cells characterized byCD93⁻ B220⁺ CD21/35^(int) IgM^(lo) IgD^(hi) expression.
 25. The mouse ofclaim 1, wherein the mouse comprises a follicular type 2 (FO-II) splenicB cell population within the range of about 1×10⁶ to about 2×10⁶ cellscharacterized by CD93⁻ B220⁺ CD21/35^(int) IgM^(int) IgD^(hi)expression.
 26. An isolated cell of the mouse of claim
 1. 27. The cellof claim 26, wherein the cell is an embryonic stem (ES) cell.
 28. Amouse embryo that comprises the ES cell of claim
 27. 29. The cell ofclaim 26, wherein the cell is a B cell.
 30. A hybridoma made with the Bcell of claim 29.